Structured Review

Illumina Inc input dna
Establishment of a high‐resolution MNase‐ChIP‐seq protocol for Trypanosoma brucei Outline of MNase‐ChIP‐seq. T. brucei cells were formaldehyde‐cross‐linked and permeabilized, and chromatin was digested into mononucleosomes using MNase. Nucleosomes containing histone H3 were isolated via affinity purification using rabbit H3 antiserum. After reversing cross‐links, the nucleosomal <t>DNA</t> was purified and paired‐end‐sequenced using <t>Illumina</t> HiSeq 2500. The sequencing reads were joined to fragments and assembled according to their midpoints. 2% agarose gel with 100 ng of mononucleosomal DNA after an MNase digest. Fragment size distribution after sequencing and joining of paired sequencing reads. Dashed lines indicate the fragment sizes 100, 137, 147, and 157 bp. Relative frequencies of AA/AT/TA/TT and CC/CG/GC/GG dinucleotides throughout 147 bp of nucleosomal DNA for each bp relative to the nucleosome dyad. Dashed lines indicate distance of 10 bp from position −74 bp.
Input Dna, supplied by Illumina Inc, used in various techniques. Bioz Stars score: 94/100, based on 224 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/result/input dna/product/Illumina Inc
Average 94 stars, based on 224 article reviews
Price from $9.99 to $1999.99
input dna - by Bioz Stars, 2020-07
94/100 stars

Images

1) Product Images from "GT‐rich promoters can drive RNA pol II transcription and deposition of H2A.Z in African trypanosomes"

Article Title: GT‐rich promoters can drive RNA pol II transcription and deposition of H2A.Z in African trypanosomes

Journal: The EMBO Journal

doi: 10.15252/embj.201695323

Establishment of a high‐resolution MNase‐ChIP‐seq protocol for Trypanosoma brucei Outline of MNase‐ChIP‐seq. T. brucei cells were formaldehyde‐cross‐linked and permeabilized, and chromatin was digested into mononucleosomes using MNase. Nucleosomes containing histone H3 were isolated via affinity purification using rabbit H3 antiserum. After reversing cross‐links, the nucleosomal DNA was purified and paired‐end‐sequenced using Illumina HiSeq 2500. The sequencing reads were joined to fragments and assembled according to their midpoints. 2% agarose gel with 100 ng of mononucleosomal DNA after an MNase digest. Fragment size distribution after sequencing and joining of paired sequencing reads. Dashed lines indicate the fragment sizes 100, 137, 147, and 157 bp. Relative frequencies of AA/AT/TA/TT and CC/CG/GC/GG dinucleotides throughout 147 bp of nucleosomal DNA for each bp relative to the nucleosome dyad. Dashed lines indicate distance of 10 bp from position −74 bp.
Figure Legend Snippet: Establishment of a high‐resolution MNase‐ChIP‐seq protocol for Trypanosoma brucei Outline of MNase‐ChIP‐seq. T. brucei cells were formaldehyde‐cross‐linked and permeabilized, and chromatin was digested into mononucleosomes using MNase. Nucleosomes containing histone H3 were isolated via affinity purification using rabbit H3 antiserum. After reversing cross‐links, the nucleosomal DNA was purified and paired‐end‐sequenced using Illumina HiSeq 2500. The sequencing reads were joined to fragments and assembled according to their midpoints. 2% agarose gel with 100 ng of mononucleosomal DNA after an MNase digest. Fragment size distribution after sequencing and joining of paired sequencing reads. Dashed lines indicate the fragment sizes 100, 137, 147, and 157 bp. Relative frequencies of AA/AT/TA/TT and CC/CG/GC/GG dinucleotides throughout 147 bp of nucleosomal DNA for each bp relative to the nucleosome dyad. Dashed lines indicate distance of 10 bp from position −74 bp.

Techniques Used: Chromatin Immunoprecipitation, Isolation, Affinity Purification, Purification, Sequencing, Agarose Gel Electrophoresis

2) Product Images from "Transposon insertion libraries for the characterization of mutants from the kiwifruit pathogen Pseudomonas syringae pv. actinidiae"

Article Title: Transposon insertion libraries for the characterization of mutants from the kiwifruit pathogen Pseudomonas syringae pv. actinidiae

Journal: PLoS ONE

doi: 10.1371/journal.pone.0172790

Preliminary characterization of Psa transposon mutants. (A) Colony size variation between wild-type Psa (left) and transposon mutants (right). A region of each plate, boxed in black, is enlarged (2×) for comparison; (B) Evaluation of the ability of transposon mutants to express GUS on KB-Km agar medium containing X-Gluc; (C) Arbitrary PCR to amplify transposon insertion sites from the genomic DNA of 32 independent transposon mutants (1–32). PCR amplicons from samples labelled in red were sequenced to characterize the specific location(s) of genome insertion by the transposon. M = DNA ladder, bp = base pairs,– = H 2 O negative control, WT = wild-type Psa genomic DNA.
Figure Legend Snippet: Preliminary characterization of Psa transposon mutants. (A) Colony size variation between wild-type Psa (left) and transposon mutants (right). A region of each plate, boxed in black, is enlarged (2×) for comparison; (B) Evaluation of the ability of transposon mutants to express GUS on KB-Km agar medium containing X-Gluc; (C) Arbitrary PCR to amplify transposon insertion sites from the genomic DNA of 32 independent transposon mutants (1–32). PCR amplicons from samples labelled in red were sequenced to characterize the specific location(s) of genome insertion by the transposon. M = DNA ladder, bp = base pairs,– = H 2 O negative control, WT = wild-type Psa genomic DNA.

Techniques Used: Polymerase Chain Reaction, Negative Control

Validation of the Psa mutant of interest (MOI) library. (A) PCR screen to identify disruptions in the IYO_023025 gene. Pooled genomic DNA samples from the columns (lanes 1–12) and rows (lanes A–H) of MOI library plate 3 (P3) were used as templates for PCR. Two sets of amplicons that share a specific IYO_023025 disruption across a single pooled column and row sample are boxed in green and red, respectively; (B) Location of the P3-G10 and P3-D4 wells, which contain a mutant with an IYO_023025 disruption specific to the PCR amplicons boxed in green and red in (A), respectively; (C) Schematic of the IYO_023025 gene showing the location of transposon insertion sites identified in (A). Transposon insertion sites are denoted by arrows, and are color-coded to match the PCR amplicons boxed green and red in (A); (D) PCR screen to identify the IYO_023025 disruption mutant from well P3-G10. Pooled genomic DNA samples from the columns and rows of a 96-well plate (P2) containing independent colony-forming units of well P3-G10 were used as templates for PCR; (E) Location of intersecting wells in P2 (dark green) that possibly contain the IYO_023025 P3-G10 disruption mutant, as determined by the PCR amplicon profile in (D); (F) PCR screen to determine which of the intersecting wells in (E) contain the IYO_023025 P3-G10 disruption mutant. Amplicons shown in the left and right panels (separated by a black line) are derived from different regions of the same gel. Wells that contain the mutant are shown in bold in (E); (G) Colony morphology of wild-type (WT) Psa and the IYO_023025 P3-G10 disruption mutant. Bar = 2 μM, M = DNA ladder, bp = base pairs,– = H 2 O negative control, WT = WT Psa DNA.
Figure Legend Snippet: Validation of the Psa mutant of interest (MOI) library. (A) PCR screen to identify disruptions in the IYO_023025 gene. Pooled genomic DNA samples from the columns (lanes 1–12) and rows (lanes A–H) of MOI library plate 3 (P3) were used as templates for PCR. Two sets of amplicons that share a specific IYO_023025 disruption across a single pooled column and row sample are boxed in green and red, respectively; (B) Location of the P3-G10 and P3-D4 wells, which contain a mutant with an IYO_023025 disruption specific to the PCR amplicons boxed in green and red in (A), respectively; (C) Schematic of the IYO_023025 gene showing the location of transposon insertion sites identified in (A). Transposon insertion sites are denoted by arrows, and are color-coded to match the PCR amplicons boxed green and red in (A); (D) PCR screen to identify the IYO_023025 disruption mutant from well P3-G10. Pooled genomic DNA samples from the columns and rows of a 96-well plate (P2) containing independent colony-forming units of well P3-G10 were used as templates for PCR; (E) Location of intersecting wells in P2 (dark green) that possibly contain the IYO_023025 P3-G10 disruption mutant, as determined by the PCR amplicon profile in (D); (F) PCR screen to determine which of the intersecting wells in (E) contain the IYO_023025 P3-G10 disruption mutant. Amplicons shown in the left and right panels (separated by a black line) are derived from different regions of the same gel. Wells that contain the mutant are shown in bold in (E); (G) Colony morphology of wild-type (WT) Psa and the IYO_023025 P3-G10 disruption mutant. Bar = 2 μM, M = DNA ladder, bp = base pairs,– = H 2 O negative control, WT = WT Psa DNA.

Techniques Used: Mutagenesis, Polymerase Chain Reaction, Amplification, Derivative Assay, Negative Control

3) Product Images from "Quantitative BrdU immunoprecipitation method demonstrates that Fkh1 and Fkh2 are rate-limiting activators of replication origins that reprogram replication timing in G1 phase"

Article Title: Quantitative BrdU immunoprecipitation method demonstrates that Fkh1 and Fkh2 are rate-limiting activators of replication origins that reprogram replication timing in G1 phase

Journal: Genome Research

doi: 10.1101/gr.196857.115

Quantitative BrdU-IP-seq analysis. ( A ) Scheme of the method. BrdU-labeled genomic DNA from each sample is barcoded by end-ligation of Illumina-compatible linkers. Samples are pooled, a small fraction of this pool is set aside as “Input,” and the remainder is subjected to immunoprecipitation (IP) with anti-BrdU antibody. The IP and Input samples are PCR-amplified with indexed primers and sequenced. IP sample reads are normalized against Input sample reads. ( B ) Validation of the method. A BrdU-labeled genomic DNA sample was split in two and each aliquot was uniquely barcoded. The distinctly barcoded samples were pooled at a ratio of 1:10 (color-keyed) and processed as above. IP results are shown raw and with normalization against the Input. ( C ) Comparison to CPM and Quantile normalization (Q-norm). JPy88 (WT) cells were synchronized in G1 phase with α-factor, released into S phase, and aliquots were incubated with BrdU for the indicated time intervals and harvested. The samples were processed as described for qBrdU-seq in A , and the IP sequence reads were analyzed by qBrdU-seq, CPM, or Quantile normalization and plotted as overlays of the time points.
Figure Legend Snippet: Quantitative BrdU-IP-seq analysis. ( A ) Scheme of the method. BrdU-labeled genomic DNA from each sample is barcoded by end-ligation of Illumina-compatible linkers. Samples are pooled, a small fraction of this pool is set aside as “Input,” and the remainder is subjected to immunoprecipitation (IP) with anti-BrdU antibody. The IP and Input samples are PCR-amplified with indexed primers and sequenced. IP sample reads are normalized against Input sample reads. ( B ) Validation of the method. A BrdU-labeled genomic DNA sample was split in two and each aliquot was uniquely barcoded. The distinctly barcoded samples were pooled at a ratio of 1:10 (color-keyed) and processed as above. IP results are shown raw and with normalization against the Input. ( C ) Comparison to CPM and Quantile normalization (Q-norm). JPy88 (WT) cells were synchronized in G1 phase with α-factor, released into S phase, and aliquots were incubated with BrdU for the indicated time intervals and harvested. The samples were processed as described for qBrdU-seq in A , and the IP sequence reads were analyzed by qBrdU-seq, CPM, or Quantile normalization and plotted as overlays of the time points.

Techniques Used: Labeling, Ligation, Immunoprecipitation, Polymerase Chain Reaction, Amplification, Incubation, Sequencing

4) Product Images from "SINE transcription by RNA polymerase III is suppressed by histone methylation but not by DNA methylation"

Article Title: SINE transcription by RNA polymerase III is suppressed by histone methylation but not by DNA methylation

Journal: Nature Communications

doi: 10.1038/ncomms7569

Pol III co-occupies methylated SINEs with MBPs. ( a ) Semiquantitative ChIP assay in A31 fibroblasts showing specific binding of TFIIIB, TFIIIC and pol III to B1 and B2 loci, as well as 7SL , but not the Apo-E gene. Histone H3 and TAF I 48 provide positive and negative controls, respectively. ( b ) Semiquantitative ChIP assay in HeLa cells showing occupancy of pol III, TFIIIB and TFIIIC at Alu loci from chromosomes 6, 10, 19 and 22, as well as 7SL and Apo-E genes. ChIPs for histone H3 and TAF I 48 provide positive and negative controls, respectively. No antibody was used for the mock sample. ( c ) Mean±s.e.m. of the percentage input bound in three independent ChIP–quantitative PCR (qPCR) assays in HeLa cells, of the indicated proteins at individual Alu loci from chromosomes 6, 10, 19 and 22, as well as 7SL and Apo-E loci and Alu PV subfamily consensus. ChIPs for histone H3 and TAF I 48 provide positive and negative controls, respectively. No antibody was used for the mock samples. P values are calculated by t -test. ( d ) Mean±s.e.m. of four independent sequential ChIP–qPCR assays in which DNA immunoprecipitated from HeLa cells using pol III antibody was reprecipitated using antibodies against pol III, TFIIIB, TAF I 48 (negative control), MBD1, MBD2 and MeCP2, as indicated. No TAF I 48 signal was detected on Alu(c6).
Figure Legend Snippet: Pol III co-occupies methylated SINEs with MBPs. ( a ) Semiquantitative ChIP assay in A31 fibroblasts showing specific binding of TFIIIB, TFIIIC and pol III to B1 and B2 loci, as well as 7SL , but not the Apo-E gene. Histone H3 and TAF I 48 provide positive and negative controls, respectively. ( b ) Semiquantitative ChIP assay in HeLa cells showing occupancy of pol III, TFIIIB and TFIIIC at Alu loci from chromosomes 6, 10, 19 and 22, as well as 7SL and Apo-E genes. ChIPs for histone H3 and TAF I 48 provide positive and negative controls, respectively. No antibody was used for the mock sample. ( c ) Mean±s.e.m. of the percentage input bound in three independent ChIP–quantitative PCR (qPCR) assays in HeLa cells, of the indicated proteins at individual Alu loci from chromosomes 6, 10, 19 and 22, as well as 7SL and Apo-E loci and Alu PV subfamily consensus. ChIPs for histone H3 and TAF I 48 provide positive and negative controls, respectively. No antibody was used for the mock samples. P values are calculated by t -test. ( d ) Mean±s.e.m. of four independent sequential ChIP–qPCR assays in which DNA immunoprecipitated from HeLa cells using pol III antibody was reprecipitated using antibodies against pol III, TFIIIB, TAF I 48 (negative control), MBD1, MBD2 and MeCP2, as indicated. No TAF I 48 signal was detected on Alu(c6).

Techniques Used: Methylation, Chromatin Immunoprecipitation, Binding Assay, Real-time Polymerase Chain Reaction, Immunoprecipitation, Negative Control

SINE expression is not stimulated by loss of DNA methylation. ( a ) Analysis by semiquantitative RT–PCR of expression levels of the indicated transcripts in matched Dnmt1 +/+ and Dnmt1 −/− fibroblasts. Duplicate samples are shown for both cell types. Apo-E and p53BP2 mRNAs provide controls that have been documented as being suppressed by DNA methylation. GAPDH mRNA provides a loading control. ( b ) Analysis by semiquantitative RT–PCR of expression levels of the indicated transcripts in mouse ES cells treated for 16 h with (+) or without (−) 5-azacytidine. Apo-E mRNA provides a control that has been documented as being inhibited by DNA methylation. ARPP P0 mRNA provides a loading control. ( c ) Analysis by semiquantitative RT–PCR of expression levels of the indicated transcripts in HeLa cells treated for 72 h with 5-azacytidine. Apo-E mRNA provides a control that has been documented as being inhibited by DNA methylation. ARPP P0 mRNA provides a loading control. ( d ) Analysis by primer extension of Alu transcripts in the RNA from Fig. 5c . Bracket indicates ~240 bp products that initiate at the principle pol III start site of Alu. Reverse transcriptase was omitted from the reactions in lanes 1 and 2. To confirm that the assay was not saturated, raising the amount of template RNA from 5 (lanes 5 and 6) to 10 μg (lanes 3 and 4) is shown to give a stronger signal. Alu, B1 and B2 RT–PCRs were performed with Alu, B1 and B2 consensus primers, respectively ( Supplementary Table 1 ).
Figure Legend Snippet: SINE expression is not stimulated by loss of DNA methylation. ( a ) Analysis by semiquantitative RT–PCR of expression levels of the indicated transcripts in matched Dnmt1 +/+ and Dnmt1 −/− fibroblasts. Duplicate samples are shown for both cell types. Apo-E and p53BP2 mRNAs provide controls that have been documented as being suppressed by DNA methylation. GAPDH mRNA provides a loading control. ( b ) Analysis by semiquantitative RT–PCR of expression levels of the indicated transcripts in mouse ES cells treated for 16 h with (+) or without (−) 5-azacytidine. Apo-E mRNA provides a control that has been documented as being inhibited by DNA methylation. ARPP P0 mRNA provides a loading control. ( c ) Analysis by semiquantitative RT–PCR of expression levels of the indicated transcripts in HeLa cells treated for 72 h with 5-azacytidine. Apo-E mRNA provides a control that has been documented as being inhibited by DNA methylation. ARPP P0 mRNA provides a loading control. ( d ) Analysis by primer extension of Alu transcripts in the RNA from Fig. 5c . Bracket indicates ~240 bp products that initiate at the principle pol III start site of Alu. Reverse transcriptase was omitted from the reactions in lanes 1 and 2. To confirm that the assay was not saturated, raising the amount of template RNA from 5 (lanes 5 and 6) to 10 μg (lanes 3 and 4) is shown to give a stronger signal. Alu, B1 and B2 RT–PCRs were performed with Alu, B1 and B2 consensus primers, respectively ( Supplementary Table 1 ).

Techniques Used: Expressing, DNA Methylation Assay, Reverse Transcription Polymerase Chain Reaction

DNA methylation does not prevent pol III occupancy of SINEs. ( a ) Percentage input bound in three independent ChIP–quantitative PCR (qPCR) assays with mouse ES cells treated for 16 h with (+) or without (−) 5-azacytidine, showing occupancy of MBD2, MeCP2 and pol III at 7SL, B1 and B2 loci, as well as an Alu inserted onto chromosomes 14 and 17. ChIPs for TAF I 48 and without antibody (mock) provide negative controls. ( b ) Percentage input bound in three independent ChIP–qPCR assays with HeLa cells treated for 72 h with (+) or without (−) 5-azacytidine, showing the binding of MBD2, TFIIIB, TFIIIC and pol III to DNA centred over the body of Alu(c22) or 200 bp downstream. The resolution of this assay is limited by the size of the genomic DNA fragments (~500 bp). ( c ) Percentage input bound in two independent ChIP–qPCR assays with matched Dnmt1 +/+ and Dnmt1 −/− fibroblasts showing occupancy of MBD2, TFIIIB, TFIIIC and pol III at B1 and B2 loci, as well as 7SL and Apo-E genes. ChIPs for TAF I 48 and without antibody (mock) provide negative controls. Error bars indicate s.e.m. and all P values are calculated by t -test.
Figure Legend Snippet: DNA methylation does not prevent pol III occupancy of SINEs. ( a ) Percentage input bound in three independent ChIP–quantitative PCR (qPCR) assays with mouse ES cells treated for 16 h with (+) or without (−) 5-azacytidine, showing occupancy of MBD2, MeCP2 and pol III at 7SL, B1 and B2 loci, as well as an Alu inserted onto chromosomes 14 and 17. ChIPs for TAF I 48 and without antibody (mock) provide negative controls. ( b ) Percentage input bound in three independent ChIP–qPCR assays with HeLa cells treated for 72 h with (+) or without (−) 5-azacytidine, showing the binding of MBD2, TFIIIB, TFIIIC and pol III to DNA centred over the body of Alu(c22) or 200 bp downstream. The resolution of this assay is limited by the size of the genomic DNA fragments (~500 bp). ( c ) Percentage input bound in two independent ChIP–qPCR assays with matched Dnmt1 +/+ and Dnmt1 −/− fibroblasts showing occupancy of MBD2, TFIIIB, TFIIIC and pol III at B1 and B2 loci, as well as 7SL and Apo-E genes. ChIPs for TAF I 48 and without antibody (mock) provide negative controls. Error bars indicate s.e.m. and all P values are calculated by t -test.

Techniques Used: DNA Methylation Assay, Chromatin Immunoprecipitation, Real-time Polymerase Chain Reaction, Binding Assay

5) Product Images from "Impact of library preparation protocols and template quantity on the metagenomic reconstruction of a mock microbial community"

Article Title: Impact of library preparation protocols and template quantity on the metagenomic reconstruction of a mock microbial community

Journal: BMC Genomics

doi: 10.1186/s12864-015-2063-6

Sample overview. Each tube on this plot represents a mock metagenomic library preparation. The control library is an unamplified TruSeq library of the same mock community sample generated from 200 ng input DNA
Figure Legend Snippet: Sample overview. Each tube on this plot represents a mock metagenomic library preparation. The control library is an unamplified TruSeq library of the same mock community sample generated from 200 ng input DNA

Techniques Used: Generated

6) Product Images from "Activation of Oncogenic Super-Enhancers Is Coupled with DNA Repair by RAD51"

Article Title: Activation of Oncogenic Super-Enhancers Is Coupled with DNA Repair by RAD51

Journal: Cell Reports

doi: 10.1016/j.celrep.2019.09.001

TOP1, but Not TOP2, Is Involved in RAD51-Associated DSBs (A) TOP2 binding (from Manville et al., 2015 ) and DSBs after ETO treatment (5 μM for 4 h) show no correlation with RAD51 binding sites at strong enhancers. (B) TOP1 ChIP-seq compared with RAD51 binding sites at strong enhancers, insulators, and promoters showing high overlap at strong enhancers. (C) TOP1 is enriched at DSBs in enhancers. DSBs are compared between 1,000 enhancers enriched with TOP1 and 1,000 enhancers exhibiting low TOP1 coverage (blue and green graphs, respectively). Rich TOP1 regions are correlated with more DSBs. (D) qPCR on genomic DNA isolated from cells treated with DMSO (mock), 5 μM B02, and 10 μM CPT for 4 h using primers designed for the sites shown in Figure S6 D. Decrease in PCR products indicates increase of DSBs, which prevents amplification. Site with no recurrent DSBs was used for normalization. Values represent mean ± SD from three independent biological samples with technical triplicates.
Figure Legend Snippet: TOP1, but Not TOP2, Is Involved in RAD51-Associated DSBs (A) TOP2 binding (from Manville et al., 2015 ) and DSBs after ETO treatment (5 μM for 4 h) show no correlation with RAD51 binding sites at strong enhancers. (B) TOP1 ChIP-seq compared with RAD51 binding sites at strong enhancers, insulators, and promoters showing high overlap at strong enhancers. (C) TOP1 is enriched at DSBs in enhancers. DSBs are compared between 1,000 enhancers enriched with TOP1 and 1,000 enhancers exhibiting low TOP1 coverage (blue and green graphs, respectively). Rich TOP1 regions are correlated with more DSBs. (D) qPCR on genomic DNA isolated from cells treated with DMSO (mock), 5 μM B02, and 10 μM CPT for 4 h using primers designed for the sites shown in Figure S6 D. Decrease in PCR products indicates increase of DSBs, which prevents amplification. Site with no recurrent DSBs was used for normalization. Values represent mean ± SD from three independent biological samples with technical triplicates.

Techniques Used: Binding Assay, Chromatin Immunoprecipitation, Real-time Polymerase Chain Reaction, Isolation, Cycling Probe Technology, Polymerase Chain Reaction, Amplification

7) Product Images from "Rational “Error Elimination” Approach to Evaluating Molecular Barcoded Next-Generation Sequencing Data Identifies Low-Frequency Mutations in Hematologic Malignancies"

Article Title: Rational “Error Elimination” Approach to Evaluating Molecular Barcoded Next-Generation Sequencing Data Identifies Low-Frequency Mutations in Hematologic Malignancies

Journal: The Journal of Molecular Diagnostics : JMD

doi: 10.1016/j.jmoldx.2019.01.008

Evaluation of molecular barcode–containing libraries by real-time quantitative PCR (qPCR) and next-generation sequencing (NGS). A: qPCR evaluation of size-selected libraries indicate uniform representation of 21 amplicons in the libraries. Note that all amplicons were present within a 2- to 3-C t value difference from the median. B: NGS indicates relatively uniform representation of the 21 amplicons in sequencing libraries. Libraries were sequenced in two independent runs, and the absolute read counts for each amplicon are depicted. C: An NGS data analysis without using molecular barcode information indicates the presence of abundant false-positive mutations at low frequencies. Libraries prepared from the reference DNA mix containing 98.4% (p.Q61L) NRAS , 1% (p.E285K) TP53 , 0.5% (p.R213Q; p.Y234H) TP53 , 0.5% (p.G13C) KRAS , and 0.1% (p.V600E) BRAF mutations were sequenced independently twice; the results of one sequencing run are shown. Note that the expected mutations above 0.3% allelic frequency were clearly apparent, although false-positive mutations appeared in this range within the amplicons covering KRAS exon 4 and NRAS exon 3. More false-positive mutations are observed between 0.05% and 0.3% allelic frequencies, and the true BRAF (V600E) mutation within this range is obscured by the false positives.
Figure Legend Snippet: Evaluation of molecular barcode–containing libraries by real-time quantitative PCR (qPCR) and next-generation sequencing (NGS). A: qPCR evaluation of size-selected libraries indicate uniform representation of 21 amplicons in the libraries. Note that all amplicons were present within a 2- to 3-C t value difference from the median. B: NGS indicates relatively uniform representation of the 21 amplicons in sequencing libraries. Libraries were sequenced in two independent runs, and the absolute read counts for each amplicon are depicted. C: An NGS data analysis without using molecular barcode information indicates the presence of abundant false-positive mutations at low frequencies. Libraries prepared from the reference DNA mix containing 98.4% (p.Q61L) NRAS , 1% (p.E285K) TP53 , 0.5% (p.R213Q; p.Y234H) TP53 , 0.5% (p.G13C) KRAS , and 0.1% (p.V600E) BRAF mutations were sequenced independently twice; the results of one sequencing run are shown. Note that the expected mutations above 0.3% allelic frequency were clearly apparent, although false-positive mutations appeared in this range within the amplicons covering KRAS exon 4 and NRAS exon 3. More false-positive mutations are observed between 0.05% and 0.3% allelic frequencies, and the true BRAF (V600E) mutation within this range is obscured by the false positives.

Techniques Used: Real-time Polymerase Chain Reaction, Next-Generation Sequencing, Sequencing, Amplification, Mutagenesis

Evaluation of three different polymerase master mixes in the first and second stages of PCR, for sequencing library preparation. Amplification reaction mixes were assembled with TaqMan genotyping master mix, HotStarTaq Plus master mix, or NEBNext Ultra II Q5 mix during first-stage PCR. All of the first-stage PCR products were assembled in NEBNext Ultra II Q5 master mix ( A ), HotStarTaq Plus master mix ( B ), or TaqMan genotyping master mix ( C ) for second-stage PCR. Libraries were purified with solid-phase reversible immobilization beads and analyzed on an Agilent 2100 DNA bioanalyzer. A 300- to 400-bp target-specific library is indicated by a bracket . Note that the fragments of 100 to 200 bp predominantly contained primer dimers. Green and purple bars indicate lower and upper markers, respectively. All samples were evaluated in triplicate.
Figure Legend Snippet: Evaluation of three different polymerase master mixes in the first and second stages of PCR, for sequencing library preparation. Amplification reaction mixes were assembled with TaqMan genotyping master mix, HotStarTaq Plus master mix, or NEBNext Ultra II Q5 mix during first-stage PCR. All of the first-stage PCR products were assembled in NEBNext Ultra II Q5 master mix ( A ), HotStarTaq Plus master mix ( B ), or TaqMan genotyping master mix ( C ) for second-stage PCR. Libraries were purified with solid-phase reversible immobilization beads and analyzed on an Agilent 2100 DNA bioanalyzer. A 300- to 400-bp target-specific library is indicated by a bracket . Note that the fragments of 100 to 200 bp predominantly contained primer dimers. Green and purple bars indicate lower and upper markers, respectively. All samples were evaluated in triplicate.

Techniques Used: Polymerase Chain Reaction, Sequencing, Amplification, Purification

Identification of parameters crucial for improving the quality of molecular barcode–containing next-generation sequencing libraries. A: Exonuclease I treatment reduces the primer dimer concentration and improves the yield of sequencing libraries. First-stage PCR products were incubated with 1 μL of 10 mmol/L Tris-Cl (pH 8.0) or exonuclease I (20 U/μL) at 37°C for 30 minutes. B: Identification of an optimal number of second-stage PCR cycles for library preparation. The first-stage PCR amplification was performed in TaqMan genotyping master mix. The products were then digested with exonuclease I. The second-stage PCR amplification with Ultra II Q5 mix was performed for 17, 20, 23, or 26 cycles. The second-stage PCR products were purified with solid-phase reversible immobilization beads and run on the Agilent 2100 DNA bioanalyzer. C: Size selection efficiently eliminated primer dimers. Genomic DNA mixes A (1% A375, 0.5% Raji, 0.1% NCI-1355, and 98.4% OCI-AML3 DNA; lanes 1, 2, 5, and 6, respectively) and B (1% NCI-1355, 0.5% Raji, 0.1% A375, and 98.4% OCI-AML3 DNA; lanes 3, 4, 7, and 8, respectively) were created and subjected to first-stage PCR amplification, exonuclease I treatment, and second-stage PCR amplification. The purified second-stage PCR products were used for double-size selection with 056×/0.85× volumes of solid-phase reversible immobilization beads, and the size-selected libraries were analyzed on the Agilent 2100 DNA bioanalyzer. Note that a 300- to 400-bp target-specific library is indicated by brackets . Green and purple bars indicate lower and upper markers, respectively. All samples were evaluated in duplicate ( B and C ) or in triplicate ( A ).
Figure Legend Snippet: Identification of parameters crucial for improving the quality of molecular barcode–containing next-generation sequencing libraries. A: Exonuclease I treatment reduces the primer dimer concentration and improves the yield of sequencing libraries. First-stage PCR products were incubated with 1 μL of 10 mmol/L Tris-Cl (pH 8.0) or exonuclease I (20 U/μL) at 37°C for 30 minutes. B: Identification of an optimal number of second-stage PCR cycles for library preparation. The first-stage PCR amplification was performed in TaqMan genotyping master mix. The products were then digested with exonuclease I. The second-stage PCR amplification with Ultra II Q5 mix was performed for 17, 20, 23, or 26 cycles. The second-stage PCR products were purified with solid-phase reversible immobilization beads and run on the Agilent 2100 DNA bioanalyzer. C: Size selection efficiently eliminated primer dimers. Genomic DNA mixes A (1% A375, 0.5% Raji, 0.1% NCI-1355, and 98.4% OCI-AML3 DNA; lanes 1, 2, 5, and 6, respectively) and B (1% NCI-1355, 0.5% Raji, 0.1% A375, and 98.4% OCI-AML3 DNA; lanes 3, 4, 7, and 8, respectively) were created and subjected to first-stage PCR amplification, exonuclease I treatment, and second-stage PCR amplification. The purified second-stage PCR products were used for double-size selection with 056×/0.85× volumes of solid-phase reversible immobilization beads, and the size-selected libraries were analyzed on the Agilent 2100 DNA bioanalyzer. Note that a 300- to 400-bp target-specific library is indicated by brackets . Green and purple bars indicate lower and upper markers, respectively. All samples were evaluated in duplicate ( B and C ) or in triplicate ( A ).

Techniques Used: Next-Generation Sequencing, Concentration Assay, Sequencing, Polymerase Chain Reaction, Incubation, Amplification, Purification, Selection

8) Product Images from "Modulation of Enhancer Looping and Differential Gene Targeting by Epstein-Barr Virus Transcription Factors Directs Cellular Reprogramming"

Article Title: Modulation of Enhancer Looping and Differential Gene Targeting by Epstein-Barr Virus Transcription Factors Directs Cellular Reprogramming

Journal: PLoS Pathogens

doi: 10.1371/journal.ppat.1003636

EBNA 2 and EBNA 3 protein binding at the WEE1 locus in EBV infected cells. (A) EBNA 2 (green) and EBNA 3 (red) sequencing reads at the WEE1 locus (displayed as described in Figure 3 ). Panels B–E show ChIP-QPCR carried out in Mutu III cells and panels F–I show data from the PER253 B95.8 LCL. Precipitated DNA was analysed using primer sets located at the binding sites (sets B, D, F, H and J) or regions adjacent to the binding sites (sets A, C, E, G and I). Binding signals at the CTBP2 binding site in the same ChIP experiments are shown as a positive control and primers spanning the transcription start site of the cellular gene PPIA provide a background binding control (indicated by dotted lines). (B) and (F) ChIP using anti-EBNA 2 antibodies. (C) and (G) ChIP using anti-EBNA 3A antibodies. (D) and (H) ChIP using anti-EBNA 3B antibodies. (E) and (I) ChIP using anti-EBNA 3C antibodies. Percentage input signals, after subtraction of no antibody controls, are shown as the mean −/+ range of two independent ChIP experiments. (J) Q-PCR analysis of WEE1 transcript levels using cDNA from BL31 parental cells and BL31 cells infected with wild-type recombinant EBV (wtBac-2 and 3), EBNA 3C knock-out EBV (3C KO-3 and 6) or EBNA 3C revertant EBV (3Crev-2 and 4). Transcript levels were normalised to GAPDH levels and expressed relative to the level in parental BL31 cells. * indicates a p-value of
Figure Legend Snippet: EBNA 2 and EBNA 3 protein binding at the WEE1 locus in EBV infected cells. (A) EBNA 2 (green) and EBNA 3 (red) sequencing reads at the WEE1 locus (displayed as described in Figure 3 ). Panels B–E show ChIP-QPCR carried out in Mutu III cells and panels F–I show data from the PER253 B95.8 LCL. Precipitated DNA was analysed using primer sets located at the binding sites (sets B, D, F, H and J) or regions adjacent to the binding sites (sets A, C, E, G and I). Binding signals at the CTBP2 binding site in the same ChIP experiments are shown as a positive control and primers spanning the transcription start site of the cellular gene PPIA provide a background binding control (indicated by dotted lines). (B) and (F) ChIP using anti-EBNA 2 antibodies. (C) and (G) ChIP using anti-EBNA 3A antibodies. (D) and (H) ChIP using anti-EBNA 3B antibodies. (E) and (I) ChIP using anti-EBNA 3C antibodies. Percentage input signals, after subtraction of no antibody controls, are shown as the mean −/+ range of two independent ChIP experiments. (J) Q-PCR analysis of WEE1 transcript levels using cDNA from BL31 parental cells and BL31 cells infected with wild-type recombinant EBV (wtBac-2 and 3), EBNA 3C knock-out EBV (3C KO-3 and 6) or EBNA 3C revertant EBV (3Crev-2 and 4). Transcript levels were normalised to GAPDH levels and expressed relative to the level in parental BL31 cells. * indicates a p-value of

Techniques Used: Protein Binding, Infection, Sequencing, Chromatin Immunoprecipitation, Real-time Polymerase Chain Reaction, Binding Assay, Positive Control, Polymerase Chain Reaction, Recombinant, Knock-Out

The influence of EBNA 3C on chromosome looping at the ADAM28/ADAMDEC1 locus. (A) Diagram (not to scale) showing the Hind III restriction fragments around the ADAM28 locus that encompass the promoter (P), the ADAM enhancer (E, located downstream of ADAM28 ) and two intervening control regions (con1 and con2). The arrow indicates the direction of transcription. (B) Chromosome conformation analysis of the ADAM28 locus in the pz1 control BJAB cell line (−) and the E3C-3 stable EBNA 3C expressing cell line (+) using primer pairs that amplify across promoter-enhancer or promoter-control ligation junctions. Positive controls show PCR amplification from control digestion and ligation reactions carried out using PCR-amplified DNA fragments encompassing the promoter, enhancer and control regions. (C) Diagram (not to scale) showing the Aci I restriction fragments around the ADAMDEC1 locus that encompass the promoter (P), the ADAM enhancer (E, located upstream of ADAMDEC1 ) and an intervening control region (con). The arrow indicates the direction of transcription. (D) Chromosome conformation analysis of the ADAMDEC1 locus in the pz1 control BJAB cell line (−) and the E3C-3 stable EBNA 3C expressing cell line (+) using primer pairs that amplify across promoter-enhancer or promoter-control ligation junctions. Positive controls show PCR amplification from control digestion and ligation reactions carried out using PCR-amplified DNA fragments encompassing the promoter, enhancer and control region.
Figure Legend Snippet: The influence of EBNA 3C on chromosome looping at the ADAM28/ADAMDEC1 locus. (A) Diagram (not to scale) showing the Hind III restriction fragments around the ADAM28 locus that encompass the promoter (P), the ADAM enhancer (E, located downstream of ADAM28 ) and two intervening control regions (con1 and con2). The arrow indicates the direction of transcription. (B) Chromosome conformation analysis of the ADAM28 locus in the pz1 control BJAB cell line (−) and the E3C-3 stable EBNA 3C expressing cell line (+) using primer pairs that amplify across promoter-enhancer or promoter-control ligation junctions. Positive controls show PCR amplification from control digestion and ligation reactions carried out using PCR-amplified DNA fragments encompassing the promoter, enhancer and control regions. (C) Diagram (not to scale) showing the Aci I restriction fragments around the ADAMDEC1 locus that encompass the promoter (P), the ADAM enhancer (E, located upstream of ADAMDEC1 ) and an intervening control region (con). The arrow indicates the direction of transcription. (D) Chromosome conformation analysis of the ADAMDEC1 locus in the pz1 control BJAB cell line (−) and the E3C-3 stable EBNA 3C expressing cell line (+) using primer pairs that amplify across promoter-enhancer or promoter-control ligation junctions. Positive controls show PCR amplification from control digestion and ligation reactions carried out using PCR-amplified DNA fragments encompassing the promoter, enhancer and control region.

Techniques Used: Expressing, Ligation, Polymerase Chain Reaction, Amplification

EBNA 3 protein binding at the ADAM28/ADAMDEC1 intergenic enhancer in EBV-infected cells. ChIP-QPCR carried out in Mutu III cells (A–C) and the PER253 B95.8 LCL (D–F). Precipitated DNA was analysed using primer sets located at the centre of the binding site (set B) or the edges of the binding site (sets A and C). Binding signals at the CTBP2 binding site in the same ChIP experiments are shown as a positive control and primers spanning the transcription start site of the cellular gene PPIA provide a background binding control (indicated by dotted lines). (A) and (D) ChIP using anti-EBNA 3A antibodies. (B) and (E) ChIP using anti-EBNA 3B antibodies. (C) and (F) ChIP using anti-EBNA 3C antibodies.
Figure Legend Snippet: EBNA 3 protein binding at the ADAM28/ADAMDEC1 intergenic enhancer in EBV-infected cells. ChIP-QPCR carried out in Mutu III cells (A–C) and the PER253 B95.8 LCL (D–F). Precipitated DNA was analysed using primer sets located at the centre of the binding site (set B) or the edges of the binding site (sets A and C). Binding signals at the CTBP2 binding site in the same ChIP experiments are shown as a positive control and primers spanning the transcription start site of the cellular gene PPIA provide a background binding control (indicated by dotted lines). (A) and (D) ChIP using anti-EBNA 3A antibodies. (B) and (E) ChIP using anti-EBNA 3B antibodies. (C) and (F) ChIP using anti-EBNA 3C antibodies.

Techniques Used: Protein Binding, Infection, Chromatin Immunoprecipitation, Real-time Polymerase Chain Reaction, Binding Assay, Positive Control

The influence of EBNA 2 and 3C on chromosome looping at the WEE1 locus. (A) Diagram (not to scale) showing the Eco R1 restriction fragments at the WEE1 locus that encompass the promoter (P), two downstream enhancers (E1 and E2) and an intervening control region (con). The arrow indicates the direction of transcription. (B) Chromosome conformation analysis in BL31 parental cells and BL31 cells infected with wild-type recombinant EBV (wtBac-2), EBNA 3C knock-out EBV (3CKO-3) or EBNA 3C revertant EBV (3Crev-4) using primer pairs that amplify across promoter-enhancer or promoter-control ligation junctio ns. Positive controls show PCR amplification from control digestion and ligation reactions carried out using PCR-amplified DNA fragments encompassing the promoter and enhancers. (C) Chromosome conformation analysis in BL31 parental cells and BL31 cells infected with wild-type recombinant EBV (wtBac-2) or EBNA 2 KO EBV. (D) Chromosome conformation capture analysis in the ER-EB 2.5 LCL expressing EBNA 2 that is active in the presence of β-estradiol (+ est) and inactive in the absence of β-estradiol (−est). (E) Model for the control of chromatin looping by EBNA 2 and 3 proteins at WEE1 . (F) Re-ChIP analysis in Mutu III cells using anti-EBNA 2 antibodies in the first round of ChIP followed by a second round of ChIP in absence of antibody or using anti-EBNA 2 or EBNA 3A, 3B or 3C antibodies. Primers at peak 5 in enhancer 2 were used for analysis. Results show mean percentage primary input −/+ range of two independent Q-PCR reactions from a representative experiment. (G) Control re-ChIP analysis using anti-EBNA 3C antibodies in the first round followed by re-precipitation in the absence of antibody or using anti-EBNA 3C antibodies.
Figure Legend Snippet: The influence of EBNA 2 and 3C on chromosome looping at the WEE1 locus. (A) Diagram (not to scale) showing the Eco R1 restriction fragments at the WEE1 locus that encompass the promoter (P), two downstream enhancers (E1 and E2) and an intervening control region (con). The arrow indicates the direction of transcription. (B) Chromosome conformation analysis in BL31 parental cells and BL31 cells infected with wild-type recombinant EBV (wtBac-2), EBNA 3C knock-out EBV (3CKO-3) or EBNA 3C revertant EBV (3Crev-4) using primer pairs that amplify across promoter-enhancer or promoter-control ligation junctio ns. Positive controls show PCR amplification from control digestion and ligation reactions carried out using PCR-amplified DNA fragments encompassing the promoter and enhancers. (C) Chromosome conformation analysis in BL31 parental cells and BL31 cells infected with wild-type recombinant EBV (wtBac-2) or EBNA 2 KO EBV. (D) Chromosome conformation capture analysis in the ER-EB 2.5 LCL expressing EBNA 2 that is active in the presence of β-estradiol (+ est) and inactive in the absence of β-estradiol (−est). (E) Model for the control of chromatin looping by EBNA 2 and 3 proteins at WEE1 . (F) Re-ChIP analysis in Mutu III cells using anti-EBNA 2 antibodies in the first round of ChIP followed by a second round of ChIP in absence of antibody or using anti-EBNA 2 or EBNA 3A, 3B or 3C antibodies. Primers at peak 5 in enhancer 2 were used for analysis. Results show mean percentage primary input −/+ range of two independent Q-PCR reactions from a representative experiment. (G) Control re-ChIP analysis using anti-EBNA 3C antibodies in the first round followed by re-precipitation in the absence of antibody or using anti-EBNA 3C antibodies.

Techniques Used: Infection, Recombinant, Knock-Out, Ligation, Polymerase Chain Reaction, Amplification, Expressing, Chromatin Immunoprecipitation

EBNA 2 and EBNA 3 protein binding at the ITGAL promoter in EBV-infected cells. (A) EBNA 2 (green) and EBNA 3 (red) sequencing reads from immunoprecipitated Mutu III DNA plotted as in Figure 3 . Panels B–E show ChIP-QPCR carried out in Mutu III cells and panels F–I show data from the PER253 B95.8 LCL. Precipitated DNA was analysed using primer sets located at the binding sites (sets B, D, and F) or regions adjacent to the binding sites (sets A, C, and E). Binding signals at the CTBP2 binding site in the same ChIP experiments are shown as a positive control and primers spanning the transcription start site of the cellular gene PPIA provide a background binding control (indicated by dotted lines). (B) and (F) ChIP using anti-EBNA 2 antibodies. (C) and (G) ChIP using anti-EBNA 3A antibodies. (D) and (H) ChIP using anti-EBNA 3B antibodies. (E) and (I) ChIP using anti-EBNA 3C antibodies. Percentage input signals, after subtraction of no antibody controls, are shown as the mean −/+ range of two independent ChIP experiments.
Figure Legend Snippet: EBNA 2 and EBNA 3 protein binding at the ITGAL promoter in EBV-infected cells. (A) EBNA 2 (green) and EBNA 3 (red) sequencing reads from immunoprecipitated Mutu III DNA plotted as in Figure 3 . Panels B–E show ChIP-QPCR carried out in Mutu III cells and panels F–I show data from the PER253 B95.8 LCL. Precipitated DNA was analysed using primer sets located at the binding sites (sets B, D, and F) or regions adjacent to the binding sites (sets A, C, and E). Binding signals at the CTBP2 binding site in the same ChIP experiments are shown as a positive control and primers spanning the transcription start site of the cellular gene PPIA provide a background binding control (indicated by dotted lines). (B) and (F) ChIP using anti-EBNA 2 antibodies. (C) and (G) ChIP using anti-EBNA 3A antibodies. (D) and (H) ChIP using anti-EBNA 3B antibodies. (E) and (I) ChIP using anti-EBNA 3C antibodies. Percentage input signals, after subtraction of no antibody controls, are shown as the mean −/+ range of two independent ChIP experiments.

Techniques Used: Protein Binding, Infection, Sequencing, Immunoprecipitation, Chromatin Immunoprecipitation, Real-time Polymerase Chain Reaction, Binding Assay, Positive Control

EBNA 3 protein binding at the BCL2L11 promoter in EBV-infected cells. (A) EBNA 3 sequencing reads from immunoprecipitated Mutu III DNA plotted as in Figure 3 . Panels B–D show ChIP-QPCR carried out in Mutu III cells and panels F–G show data from the PER253 B95.8 LCL. Precipitated DNA was analysed using primer sets located at the binding site (set B) or regions on either side of the binding site (sets A and C). Binding signals at the CTBP2 binding site in the same ChIP experiments are shown as a positive control and primers spanning the transcription start site of the cellular gene PPIA provide a background binding control (indicated by dotted lines). (B) and (E) ChIP using anti-EBNA 3A antibodies. (C) and (F) ChIP using anti-EBNA 3B antibodies. (D) and (G) ChIP using anti-EBNA 3C antibodies. Percentage input signals, after subtraction of no antibody controls, are shown as the mean −/+ range of two independent experiments.
Figure Legend Snippet: EBNA 3 protein binding at the BCL2L11 promoter in EBV-infected cells. (A) EBNA 3 sequencing reads from immunoprecipitated Mutu III DNA plotted as in Figure 3 . Panels B–D show ChIP-QPCR carried out in Mutu III cells and panels F–G show data from the PER253 B95.8 LCL. Precipitated DNA was analysed using primer sets located at the binding site (set B) or regions on either side of the binding site (sets A and C). Binding signals at the CTBP2 binding site in the same ChIP experiments are shown as a positive control and primers spanning the transcription start site of the cellular gene PPIA provide a background binding control (indicated by dotted lines). (B) and (E) ChIP using anti-EBNA 3A antibodies. (C) and (F) ChIP using anti-EBNA 3B antibodies. (D) and (G) ChIP using anti-EBNA 3C antibodies. Percentage input signals, after subtraction of no antibody controls, are shown as the mean −/+ range of two independent experiments.

Techniques Used: Protein Binding, Infection, Sequencing, Immunoprecipitation, Chromatin Immunoprecipitation, Real-time Polymerase Chain Reaction, Binding Assay, Positive Control

EBNA 2, 3A, 3B and 3C binding at the CTBP2 locus in EBV-infected cells. (A) The number of EBNA 2 (green) and EBNA 3 (red) sequencing reads from immunoprecipitated Mutu III DNA are plotted per million background-subtracted total reads and aligned with the human genome. The direction of gene transcription is indicated by the red arrow. GM12878 LCL H3K27ac ChIP-seq data from ENCODE are shown at the bottom of the panel. Panels B-E show ChIP-QPCR carried out in Mutu III cells and panels F-I show data from the PER253 B95.8 LCL. Precipitated DNA was analysed using primer sets located at the binding site (set B) or regions on either side of the binding site (sets A and C). Primers spanning the transcription start site of the cellular gene encoding peptidylprolyl isomerase A (PPIA) that is not regulated or bound by the EBNAs provide a background binding control (indicated by dotted lines). (B) and (F) ChIP using anti-EBNA 2 antibodies. (C) and (G) ChIP using anti-EBNA 3A antibodies. (D) and (H) ChIP using anti-EBNA 3B antibodies. (E) and (I) ChIP using anti-EBNA 3C antibodies. Percentage input signals, after subtraction of no antibody controls, are shown as the mean +/− range of two independent experiments. (J) Q-PCR analysis of CTBP2 transcript levels using cDNA from wild-type LCLs (wt1, 2 and 3) and LCLs established from EBNA 3A knock-out viruses (mtB1, B2 and B3) in two different donor backgrounds (D2 and D3). Transcript levels were normalised to GAPDH levels and expressed relative to the level in D2 wt1 cells. (K) Q-PCR analysis of CTBP2 transcript levels using cDNA from wild-type LCLs infected with B95.8 virus (wt) and EBNA 3B knock-out LCLs (KO) in two different donor backgrounds (PER142 and PER253). Transcript levels were normalised to GAPDH levels and expressed relative to the level in wt cells for each donor. (L) Q-PCR analysis of CTBP2 transcript levels using cDNA from the ER/EB 2.5 LCL expressing EBNA 2 that is active in the presence of β-estradiol (+ est) and inactive in the absence of β-estradiol (−est). Cells were incubated in β-estradiol-free media for 4 days prior to re-addition of β-estradiol or DMSO control for 6 or 17 hrs. Transcript levels were normalised to GAPDH levels and expressed relative to the level in the absence of β-estradiol for each time course. All cDNA results (J–L) show the mean −/+ range of two independent QPCR reactions each performed in duplicate.
Figure Legend Snippet: EBNA 2, 3A, 3B and 3C binding at the CTBP2 locus in EBV-infected cells. (A) The number of EBNA 2 (green) and EBNA 3 (red) sequencing reads from immunoprecipitated Mutu III DNA are plotted per million background-subtracted total reads and aligned with the human genome. The direction of gene transcription is indicated by the red arrow. GM12878 LCL H3K27ac ChIP-seq data from ENCODE are shown at the bottom of the panel. Panels B-E show ChIP-QPCR carried out in Mutu III cells and panels F-I show data from the PER253 B95.8 LCL. Precipitated DNA was analysed using primer sets located at the binding site (set B) or regions on either side of the binding site (sets A and C). Primers spanning the transcription start site of the cellular gene encoding peptidylprolyl isomerase A (PPIA) that is not regulated or bound by the EBNAs provide a background binding control (indicated by dotted lines). (B) and (F) ChIP using anti-EBNA 2 antibodies. (C) and (G) ChIP using anti-EBNA 3A antibodies. (D) and (H) ChIP using anti-EBNA 3B antibodies. (E) and (I) ChIP using anti-EBNA 3C antibodies. Percentage input signals, after subtraction of no antibody controls, are shown as the mean +/− range of two independent experiments. (J) Q-PCR analysis of CTBP2 transcript levels using cDNA from wild-type LCLs (wt1, 2 and 3) and LCLs established from EBNA 3A knock-out viruses (mtB1, B2 and B3) in two different donor backgrounds (D2 and D3). Transcript levels were normalised to GAPDH levels and expressed relative to the level in D2 wt1 cells. (K) Q-PCR analysis of CTBP2 transcript levels using cDNA from wild-type LCLs infected with B95.8 virus (wt) and EBNA 3B knock-out LCLs (KO) in two different donor backgrounds (PER142 and PER253). Transcript levels were normalised to GAPDH levels and expressed relative to the level in wt cells for each donor. (L) Q-PCR analysis of CTBP2 transcript levels using cDNA from the ER/EB 2.5 LCL expressing EBNA 2 that is active in the presence of β-estradiol (+ est) and inactive in the absence of β-estradiol (−est). Cells were incubated in β-estradiol-free media for 4 days prior to re-addition of β-estradiol or DMSO control for 6 or 17 hrs. Transcript levels were normalised to GAPDH levels and expressed relative to the level in the absence of β-estradiol for each time course. All cDNA results (J–L) show the mean −/+ range of two independent QPCR reactions each performed in duplicate.

Techniques Used: Binding Assay, Infection, Sequencing, Immunoprecipitation, Chromatin Immunoprecipitation, Real-time Polymerase Chain Reaction, Polymerase Chain Reaction, Knock-Out, Expressing, Incubation

The influence of EBNA 2 and 3A on chromosome looping at the CTBP2 locus. (A) Diagram (not to scale) showing the Eco R1 restriction fragments at the CTBP2 locus that encompass the promoter (P), enhancer (E) and an intervening control region (con). The arrow indicates the direction of transcription. (B) Chromosome conformation analysis in LCLs infected with wild-type or EBNA 3A knock-out EBV using primer pairs that amplify across promoter-enhancer or promoter-control ligation junctions. Positive controls show PCR amplification from control digestion and ligation reactions carried out using PCR-amplified DNA fragments encompassing the promoter, enhancer and control regions. (C). Chromosome conformation capture analysis in the ER-EB 2.5 LCL expressing EBNA 2 that is active in the presence of β-estradiol (+ est) and inactive in the absence of β-estradiol (−est). (D) Model for the control of chromatin looping by EBNA 2 and 3 proteins at CTBP2 . (E) Re-ChIP analysis using anti-EBNA 2 antibodies in the first round of ChIP followed by a second round of ChIP in absence of antibody or using anti-EBNA 2, EBNA 3A, EBNA 3B or EBNA 3C antibodies. Results show mean percentage primary input −/+ range of two independent Q-PCR reactions from a representative experiment. (F) Control re-ChIP analysis using anti-EBNA 3A antibodies in the first round followed by re-precipitation in the absence of antibody or using anti-EBNA 3A antibodies. (G) Control re-ChIP analysis using anti-EBNA 3B antibodies in the first round followed by re-precipitation in the absence of antibody or using anti-EBNA 3B antibodies. (H) Control re-ChIP analysis using anti-EBNA 3C antibodies in the first round followed by re-precipitation in the absence of antibody or using anti-EBNA 3C antibodies.
Figure Legend Snippet: The influence of EBNA 2 and 3A on chromosome looping at the CTBP2 locus. (A) Diagram (not to scale) showing the Eco R1 restriction fragments at the CTBP2 locus that encompass the promoter (P), enhancer (E) and an intervening control region (con). The arrow indicates the direction of transcription. (B) Chromosome conformation analysis in LCLs infected with wild-type or EBNA 3A knock-out EBV using primer pairs that amplify across promoter-enhancer or promoter-control ligation junctions. Positive controls show PCR amplification from control digestion and ligation reactions carried out using PCR-amplified DNA fragments encompassing the promoter, enhancer and control regions. (C). Chromosome conformation capture analysis in the ER-EB 2.5 LCL expressing EBNA 2 that is active in the presence of β-estradiol (+ est) and inactive in the absence of β-estradiol (−est). (D) Model for the control of chromatin looping by EBNA 2 and 3 proteins at CTBP2 . (E) Re-ChIP analysis using anti-EBNA 2 antibodies in the first round of ChIP followed by a second round of ChIP in absence of antibody or using anti-EBNA 2, EBNA 3A, EBNA 3B or EBNA 3C antibodies. Results show mean percentage primary input −/+ range of two independent Q-PCR reactions from a representative experiment. (F) Control re-ChIP analysis using anti-EBNA 3A antibodies in the first round followed by re-precipitation in the absence of antibody or using anti-EBNA 3A antibodies. (G) Control re-ChIP analysis using anti-EBNA 3B antibodies in the first round followed by re-precipitation in the absence of antibody or using anti-EBNA 3B antibodies. (H) Control re-ChIP analysis using anti-EBNA 3C antibodies in the first round followed by re-precipitation in the absence of antibody or using anti-EBNA 3C antibodies.

Techniques Used: Infection, Knock-Out, Ligation, Polymerase Chain Reaction, Amplification, Expressing, Chromatin Immunoprecipitation

9) Product Images from "Efficient yeast ChIP-Seq using multiplex short-read DNA sequencing"

Article Title: Efficient yeast ChIP-Seq using multiplex short-read DNA sequencing

Journal: BMC Genomics

doi: 10.1186/1471-2164-10-37

PolII signal profiles recapitulate findings from Steinmetz et al . PolII ChIP-Seq signal profiles resemble very closely to those published in Figure 3 of Steinmetz et al [ 54 ]. We obtained consistent binding at the Bap2-Tat1 loci (a) and at the Sed1-Shu2 loci (b). As expected, we did not observe binding at the Flo11 locus (c). For PolII ChIP-Seq experiments, two biological replicates were barcoded with ACGT (PolII_Rep1, dark blue; PolII_Rep2, orange), one was barcoded with TGCT (PolII_Rep3, red) and a fourth replicate had non-barcoded adapters (PolII_Rep4, green). Input DNA serves as a reference (light blue). Axis and scale normalizations are similar to Figure 2 . ORFs above the coordinates axis are on the Watson strand while ORFs below this axis are on the Crick strand.
Figure Legend Snippet: PolII signal profiles recapitulate findings from Steinmetz et al . PolII ChIP-Seq signal profiles resemble very closely to those published in Figure 3 of Steinmetz et al [ 54 ]. We obtained consistent binding at the Bap2-Tat1 loci (a) and at the Sed1-Shu2 loci (b). As expected, we did not observe binding at the Flo11 locus (c). For PolII ChIP-Seq experiments, two biological replicates were barcoded with ACGT (PolII_Rep1, dark blue; PolII_Rep2, orange), one was barcoded with TGCT (PolII_Rep3, red) and a fourth replicate had non-barcoded adapters (PolII_Rep4, green). Input DNA serves as a reference (light blue). Axis and scale normalizations are similar to Figure 2 . ORFs above the coordinates axis are on the Watson strand while ORFs below this axis are on the Crick strand.

Techniques Used: Chromatin Immunoprecipitation, Binding Assay

Comparison of input DNA signal tracks among all four barcoded adapters relative to standard Illumina adapters . An input sample was split in five aliquots. Four were barcoded differentially (top four lanes) and one had non-barcoded, Illumina adapters (fifth lane, labeled 'None'). Barcoded inputs were scored against non-barcoded input. IGB signal tracks of yeast chromosome 16 are shown for each sample, with ORF locations on the x-axis. ORFs are depicted in purple. On the y-axis, a normalized scale represents the number of read counts at a particular location. Each scale is normalized according to the number of mapped reads (Table 10 ). A box in the left panel depicts the enlarged section shown in the right panel for positions between 828,000 and 833,000 to demonstrate the overlap among all signal tracks.
Figure Legend Snippet: Comparison of input DNA signal tracks among all four barcoded adapters relative to standard Illumina adapters . An input sample was split in five aliquots. Four were barcoded differentially (top four lanes) and one had non-barcoded, Illumina adapters (fifth lane, labeled 'None'). Barcoded inputs were scored against non-barcoded input. IGB signal tracks of yeast chromosome 16 are shown for each sample, with ORF locations on the x-axis. ORFs are depicted in purple. On the y-axis, a normalized scale represents the number of read counts at a particular location. Each scale is normalized according to the number of mapped reads (Table 10 ). A box in the left panel depicts the enlarged section shown in the right panel for positions between 828,000 and 833,000 to demonstrate the overlap among all signal tracks.

Techniques Used: Labeling

Barcoded adapters perform similarly to standard Illumina adapters and do not crossover to other samples in the same lane . (a) RNA PolII binding profiles from different biological replicates with the same barcode (PolII_Rep1, dark blue; PolII_Rep3, red), with different barcodes (PolII_Rep2, orange) or without barcode (PolII_Rep4, green) strongly overlap. See also Table 3 . Input DNA serves as a reference (light blue). IGB signal tracks of chromosome 5 between 130,000 and 320,000 are shown for each library. A box in the left panel depicts the enlarged section shown in the right panel between positions 298,000 and 309,000 to illustrate the overlap among all PolII signal tracks. (b) Binding profiles from four different libraries pooled and sequenced in the same flowcell lane show very little resemblance. Shown here are the binding profiles for Cse4_Rep2 (dark blue), Ste12_Rep2 (red), PolII_Rep2 (green) and Input_ACGT (light blue). IGB signal tracks of chromosome 12 between 80,000 and 210,000 are shown for each sample. For (a) and (b), axis and scale normalizations are similar to Figure 2 . (c) Left: Rank-rank comparison of target lists between all pairwise barcoded replicates for Cse4, PolII and Ste12. The horizontal axis shows the fraction of the two lists being compared and the vertical axis shows the fraction of those targets that agree between a given pair of target lists. All comparisons show strong agreement, although the rank lists for Cse4 differ more than PolII or Ste12 for the second half of their length. Right: Rank-rank comparison between barcoded replicates from the same factors (averaged over all pairwise comparisons) compared to rank-rank comparisons for barcoded replicates between different factors: PolII_Rep1 (ACGT) vs. Ste12_Rep1 (TGCT) and Cse4_Rep2 (CATT) vs. Ste12_Rep2 (GTAT).
Figure Legend Snippet: Barcoded adapters perform similarly to standard Illumina adapters and do not crossover to other samples in the same lane . (a) RNA PolII binding profiles from different biological replicates with the same barcode (PolII_Rep1, dark blue; PolII_Rep3, red), with different barcodes (PolII_Rep2, orange) or without barcode (PolII_Rep4, green) strongly overlap. See also Table 3 . Input DNA serves as a reference (light blue). IGB signal tracks of chromosome 5 between 130,000 and 320,000 are shown for each library. A box in the left panel depicts the enlarged section shown in the right panel between positions 298,000 and 309,000 to illustrate the overlap among all PolII signal tracks. (b) Binding profiles from four different libraries pooled and sequenced in the same flowcell lane show very little resemblance. Shown here are the binding profiles for Cse4_Rep2 (dark blue), Ste12_Rep2 (red), PolII_Rep2 (green) and Input_ACGT (light blue). IGB signal tracks of chromosome 12 between 80,000 and 210,000 are shown for each sample. For (a) and (b), axis and scale normalizations are similar to Figure 2 . (c) Left: Rank-rank comparison of target lists between all pairwise barcoded replicates for Cse4, PolII and Ste12. The horizontal axis shows the fraction of the two lists being compared and the vertical axis shows the fraction of those targets that agree between a given pair of target lists. All comparisons show strong agreement, although the rank lists for Cse4 differ more than PolII or Ste12 for the second half of their length. Right: Rank-rank comparison between barcoded replicates from the same factors (averaged over all pairwise comparisons) compared to rank-rank comparisons for barcoded replicates between different factors: PolII_Rep1 (ACGT) vs. Ste12_Rep1 (TGCT) and Cse4_Rep2 (CATT) vs. Ste12_Rep2 (GTAT).

Techniques Used: Binding Assay

Ste12 distribution during pseudohyphal growth is similar across three different biological replicates . Two barcoded replicates (Ste12_Rep2, dark blue; Ste12_Rep1, red) and a non-barcoded replicate (Ste12_Rep3, green) were compared to input DNA (light blue). Ste12 ChIP samples were scored against a pool of input DNA. IGB signal tracks of chromosome 2 between 340,000 and 410,000 are shown for each sample. Axis and scale normalizations are similar to Figure 2 . A box in the left panel containing the TEC1 gene and its surrounding intergenic region was enlarged in panel B and rescaled to emphasize the strong signal at the TEC1 promoter. The same normalization as in Figure 2 was applied. Ste12p and Tec1p act as a dimer during pseudohyphal growth [ 31 ].
Figure Legend Snippet: Ste12 distribution during pseudohyphal growth is similar across three different biological replicates . Two barcoded replicates (Ste12_Rep2, dark blue; Ste12_Rep1, red) and a non-barcoded replicate (Ste12_Rep3, green) were compared to input DNA (light blue). Ste12 ChIP samples were scored against a pool of input DNA. IGB signal tracks of chromosome 2 between 340,000 and 410,000 are shown for each sample. Axis and scale normalizations are similar to Figure 2 . A box in the left panel containing the TEC1 gene and its surrounding intergenic region was enlarged in panel B and rescaled to emphasize the strong signal at the TEC1 promoter. The same normalization as in Figure 2 was applied. Ste12p and Tec1p act as a dimer during pseudohyphal growth [ 31 ].

Techniques Used: Chromatin Immunoprecipitation, Activated Clotting Time Assay

Cse4p is found robustly at centromeres . All biological replicates were strongly and tightly bound to centromeres, as it is depicted here in the case of CEN11 . Two barcoded replicates (Cse4_Rep2, dark blue; Cse4_Rep1, red) and a non-barcoded replicate (Cse4_Rep3, green) were compared to input DNA (light blue). Cse4 ChIP samples were scored against a pool of input DNA. IGB signal tracks of the CEN11 on chromosome 11 are shown for each sample. CEN11 is highlighted in a yellow box. Axis and scale normalizations are similar to Figure 2 .
Figure Legend Snippet: Cse4p is found robustly at centromeres . All biological replicates were strongly and tightly bound to centromeres, as it is depicted here in the case of CEN11 . Two barcoded replicates (Cse4_Rep2, dark blue; Cse4_Rep1, red) and a non-barcoded replicate (Cse4_Rep3, green) were compared to input DNA (light blue). Cse4 ChIP samples were scored against a pool of input DNA. IGB signal tracks of the CEN11 on chromosome 11 are shown for each sample. CEN11 is highlighted in a yellow box. Axis and scale normalizations are similar to Figure 2 .

Techniques Used: Chromatin Immunoprecipitation

Scheme for yeast barcoded ChIP-Seq . (a) Barcoded ChIP-Seq workflow. Ovals depict yeast cells and squares depict proteins. An aliquot of sheared cell lysate is not immunoprecipitated but is otherwise processed normally (green). This DNA, termed input DNA, is a reference sample for ChIP-Seq. Illumina DNA libraries are generated from both ChIP and input DNA samples. In multiplex ChIP-Seq, a barcoded adapter is ligated to an individual DNA sample. The barcode has 3 unique bases followed by a 'T' to anneal with the end-repaired DNA. Four libraries are then pooled together and applied to a single flowcell lane. After sequencing on the Genome Analyzer, reads are separated according to the first four bases and aligned to the yeast genome. Reads are stacked to generate a signal profile and scored against a pool of input DNA to determine significant transcription factor binding sites. (b) The barcode (orange) is located between Illumina adapter sequences (purple) and ChIP or input DNA inserts (black). The sequencing primer (pink) anneals to the adapter sequences and short reads start with the four bases of the barcode (orange) followed by DNA inserts (black). For the sequencing primer and Illumina adapter, oligonucleotide sequences were given by the manufacturer © 2006 Illumina, Inc. All rights reserved.
Figure Legend Snippet: Scheme for yeast barcoded ChIP-Seq . (a) Barcoded ChIP-Seq workflow. Ovals depict yeast cells and squares depict proteins. An aliquot of sheared cell lysate is not immunoprecipitated but is otherwise processed normally (green). This DNA, termed input DNA, is a reference sample for ChIP-Seq. Illumina DNA libraries are generated from both ChIP and input DNA samples. In multiplex ChIP-Seq, a barcoded adapter is ligated to an individual DNA sample. The barcode has 3 unique bases followed by a 'T' to anneal with the end-repaired DNA. Four libraries are then pooled together and applied to a single flowcell lane. After sequencing on the Genome Analyzer, reads are separated according to the first four bases and aligned to the yeast genome. Reads are stacked to generate a signal profile and scored against a pool of input DNA to determine significant transcription factor binding sites. (b) The barcode (orange) is located between Illumina adapter sequences (purple) and ChIP or input DNA inserts (black). The sequencing primer (pink) anneals to the adapter sequences and short reads start with the four bases of the barcode (orange) followed by DNA inserts (black). For the sequencing primer and Illumina adapter, oligonucleotide sequences were given by the manufacturer © 2006 Illumina, Inc. All rights reserved.

Techniques Used: Chromatin Immunoprecipitation, Immunoprecipitation, Generated, Multiplex Assay, Sequencing, Binding Assay

10) Product Images from "Discovery of a new predominant cytosine DNA modification that is linked to gene expression in malaria parasites"

Article Title: Discovery of a new predominant cytosine DNA modification that is linked to gene expression in malaria parasites

Journal: Nucleic Acids Research

doi: 10.1093/nar/gkz1093

hmeDIP-seq performs robustly for the AT-rich Plasmodium falciparum genome. ( A ) Schematic representation of the hmeDIP-seq methodology used to measure 5hmC (or 5hmC-like) distribution in P. falciparum asexual stages. Sheared P. falciparum genomic DNA was immunoprecipitated using anti-5hmC antibodies and the resulting DNA processed for Next Generation Sequencing (NGS). MACS2 was used for 5hmC-like peak-calling. See Materials and Methods for additional details. ( B ) Correlation between hmeDIP-seq biological replicates (calculated for bam alignment files derived from Illumina sequencing data) for the Ring (R), Trophozoite (Troph or T) and Schizont (Schiz or S) stages of 3D7-G7 was determined using a Pearson correlation analysis. The colour scale indicates values of the Pearson correlation coefficient R from 0 to 1. Ctrl = Control IP for biological replicate 1. A and B are technical replicates for the same genomic DNA sample. ( C ) Correlation of MACS2 fold enrichment (FE) profiles of hmeDIP-seq samples relative to Input DNA for the Ring (R), Trophozoite (T) and Schizont (S) stages of 3D7-G7 was compared to the IgG control IPs relative to Input DNA. For each stage, data were normalized across all replicates to generate a single FE profile (described in ‘Materials and Methods’ section). The colour scale indicates values of the Pearson correlation coefficient R from 0 to 1. Ctrl = Control IP for biological replicate 1. ( D ) Principal Component Analysis of the different MACS2-derived FE profiles of part C was performed using the plotPCA function of DeepTools on a multibigwigsummary file. The eigenvalues of the top two principal components PC1 (54.3% of variance explained) and PC2 (31.3% of variance explained) are shown and meaningful clustering of samples indicated.
Figure Legend Snippet: hmeDIP-seq performs robustly for the AT-rich Plasmodium falciparum genome. ( A ) Schematic representation of the hmeDIP-seq methodology used to measure 5hmC (or 5hmC-like) distribution in P. falciparum asexual stages. Sheared P. falciparum genomic DNA was immunoprecipitated using anti-5hmC antibodies and the resulting DNA processed for Next Generation Sequencing (NGS). MACS2 was used for 5hmC-like peak-calling. See Materials and Methods for additional details. ( B ) Correlation between hmeDIP-seq biological replicates (calculated for bam alignment files derived from Illumina sequencing data) for the Ring (R), Trophozoite (Troph or T) and Schizont (Schiz or S) stages of 3D7-G7 was determined using a Pearson correlation analysis. The colour scale indicates values of the Pearson correlation coefficient R from 0 to 1. Ctrl = Control IP for biological replicate 1. A and B are technical replicates for the same genomic DNA sample. ( C ) Correlation of MACS2 fold enrichment (FE) profiles of hmeDIP-seq samples relative to Input DNA for the Ring (R), Trophozoite (T) and Schizont (S) stages of 3D7-G7 was compared to the IgG control IPs relative to Input DNA. For each stage, data were normalized across all replicates to generate a single FE profile (described in ‘Materials and Methods’ section). The colour scale indicates values of the Pearson correlation coefficient R from 0 to 1. Ctrl = Control IP for biological replicate 1. ( D ) Principal Component Analysis of the different MACS2-derived FE profiles of part C was performed using the plotPCA function of DeepTools on a multibigwigsummary file. The eigenvalues of the top two principal components PC1 (54.3% of variance explained) and PC2 (31.3% of variance explained) are shown and meaningful clustering of samples indicated.

Techniques Used: Immunoprecipitation, Next-Generation Sequencing, Derivative Assay, Sequencing

11) Product Images from "Division of Labor between PCNA Loaders in DNA Replication and Sister Chromatid Cohesion Establishment"

Article Title: Division of Labor between PCNA Loaders in DNA Replication and Sister Chromatid Cohesion Establishment

Journal: Molecular Cell

doi: 10.1016/j.molcel.2020.03.017

Ctf18 and Rfc1 Distribute to the Leading and Lagging Strands (A) Schematic of eSPAN, combining ChIP with strand-specific nascent DNA sequencing. (B) Cells were synchronized in G1 and released into medium containing BrdU and HU. DNA recovered by BrdU-IP, ChIP against Ctf18 or Rfc1, and ChIP followed by BrdU-IP (eSPAN) was subject to strand-specific sequencing. Watson (red) and Crick (green) reads around ARS508 are shown, as well as the averaged strand bias, normalized to BrdU reads, surrounding 92 early, well-separated origins. (C) Ctf18 and Rfc1 eSPAN analysis in cells synchronized in G1 and released into synchronous S phase progression in BrdU-containing medium for 26 min. (D) Ctf18 eSPAN analysis in Pol2 EDD cells synchronized in G1 and released into BrdU- and HU-containing medium. See Figure S6 for Ctf18 and Rfc1 eSPAN analyses in cells lacking their respective counterparts.
Figure Legend Snippet: Ctf18 and Rfc1 Distribute to the Leading and Lagging Strands (A) Schematic of eSPAN, combining ChIP with strand-specific nascent DNA sequencing. (B) Cells were synchronized in G1 and released into medium containing BrdU and HU. DNA recovered by BrdU-IP, ChIP against Ctf18 or Rfc1, and ChIP followed by BrdU-IP (eSPAN) was subject to strand-specific sequencing. Watson (red) and Crick (green) reads around ARS508 are shown, as well as the averaged strand bias, normalized to BrdU reads, surrounding 92 early, well-separated origins. (C) Ctf18 and Rfc1 eSPAN analysis in cells synchronized in G1 and released into synchronous S phase progression in BrdU-containing medium for 26 min. (D) Ctf18 eSPAN analysis in Pol2 EDD cells synchronized in G1 and released into BrdU- and HU-containing medium. See Figure S6 for Ctf18 and Rfc1 eSPAN analyses in cells lacking their respective counterparts.

Techniques Used: Chromatin Immunoprecipitation, DNA Sequencing, Sequencing

Rfc1-RFC Promotes DNA Replication but Not Sister Chromatid Cohesion (A) Cells were synchronized in G1, and Rfc1 was depleted for 2 h by auxin treatment before release into synchronous progression through S phase. DNA replication was monitored by FACS analysis of DNA content. (B) Cells were synchronized in G1 and released into BrdU-containing medium. Cells were harvested at the indicated times, and BrdU immunoprecipitates were hybridized to Affymetrix GeneChip S. cerevisiae Tiling 1.0R arrays. Signal intensities, relative to a whole-genome DNA sample, normalized to the median BrdU peak intensities, are shown along chromosome 8. Origin positions are indicated. (C) Boxplots of BrdU peak widths derived from (B) from 52 early origins at the indicated time points. (D) Cells were arrested in G1, and Rfc1 was depleted for 2 h by auxin treatment before release into HU-containing medium for an early S phase arrest. FLAG-PCNA chromatin immunoprecipitates were analyzed by qPCR with primer pairs around ARS607. Means ± SE from four independent experiments are shown. (E) Cells were synchronized in G1 and released into nocodazole-containing medium. Sister chromatid cohesion was assessed at the GFP-marked URA3 locus. Means ± SE from three independent experiments are shown. (F) As in (E), but Smc3 acetylation was quantified relative to total Smc3 levels. Means ± SE from three independent experiments are shown. See Figure S4 for further characterization of the rfc1-aid strain.
Figure Legend Snippet: Rfc1-RFC Promotes DNA Replication but Not Sister Chromatid Cohesion (A) Cells were synchronized in G1, and Rfc1 was depleted for 2 h by auxin treatment before release into synchronous progression through S phase. DNA replication was monitored by FACS analysis of DNA content. (B) Cells were synchronized in G1 and released into BrdU-containing medium. Cells were harvested at the indicated times, and BrdU immunoprecipitates were hybridized to Affymetrix GeneChip S. cerevisiae Tiling 1.0R arrays. Signal intensities, relative to a whole-genome DNA sample, normalized to the median BrdU peak intensities, are shown along chromosome 8. Origin positions are indicated. (C) Boxplots of BrdU peak widths derived from (B) from 52 early origins at the indicated time points. (D) Cells were arrested in G1, and Rfc1 was depleted for 2 h by auxin treatment before release into HU-containing medium for an early S phase arrest. FLAG-PCNA chromatin immunoprecipitates were analyzed by qPCR with primer pairs around ARS607. Means ± SE from four independent experiments are shown. (E) Cells were synchronized in G1 and released into nocodazole-containing medium. Sister chromatid cohesion was assessed at the GFP-marked URA3 locus. Means ± SE from three independent experiments are shown. (F) As in (E), but Smc3 acetylation was quantified relative to total Smc3 levels. Means ± SE from three independent experiments are shown. See Figure S4 for further characterization of the rfc1-aid strain.

Techniques Used: FACS, Derivative Assay, Real-time Polymerase Chain Reaction

12) Product Images from "Efficient yeast ChIP-Seq using multiplex short-read DNA sequencing"

Article Title: Efficient yeast ChIP-Seq using multiplex short-read DNA sequencing

Journal: BMC Genomics

doi: 10.1186/1471-2164-10-37

Comparison of input DNA signal tracks among all four barcoded adapters relative to standard Illumina adapters . An input sample was split in five aliquots. Four were barcoded differentially (top four lanes) and one had non-barcoded, Illumina adapters (fifth lane, labeled 'None'). Barcoded inputs were scored against non-barcoded input. IGB signal tracks of yeast chromosome 16 are shown for each sample, with ORF locations on the x-axis. ORFs are depicted in purple. On the y-axis, a normalized scale represents the number of read counts at a particular location. Each scale is normalized according to the number of mapped reads (Table 10 ). A box in the left panel depicts the enlarged section shown in the right panel for positions between 828,000 and 833,000 to demonstrate the overlap among all signal tracks.
Figure Legend Snippet: Comparison of input DNA signal tracks among all four barcoded adapters relative to standard Illumina adapters . An input sample was split in five aliquots. Four were barcoded differentially (top four lanes) and one had non-barcoded, Illumina adapters (fifth lane, labeled 'None'). Barcoded inputs were scored against non-barcoded input. IGB signal tracks of yeast chromosome 16 are shown for each sample, with ORF locations on the x-axis. ORFs are depicted in purple. On the y-axis, a normalized scale represents the number of read counts at a particular location. Each scale is normalized according to the number of mapped reads (Table 10 ). A box in the left panel depicts the enlarged section shown in the right panel for positions between 828,000 and 833,000 to demonstrate the overlap among all signal tracks.

Techniques Used: Labeling

Barcoded adapters perform similarly to standard Illumina adapters and do not crossover to other samples in the same lane . (a) RNA PolII binding profiles from different biological replicates with the same barcode (PolII_Rep1, dark blue; PolII_Rep3, red), with different barcodes (PolII_Rep2, orange) or without barcode (PolII_Rep4, green) strongly overlap. See also Table 3 . Input DNA serves as a reference (light blue). IGB signal tracks of chromosome 5 between 130,000 and 320,000 are shown for each library. A box in the left panel depicts the enlarged section shown in the right panel between positions 298,000 and 309,000 to illustrate the overlap among all PolII signal tracks. (b) Binding profiles from four different libraries pooled and sequenced in the same flowcell lane show very little resemblance. Shown here are the binding profiles for Cse4_Rep2 (dark blue), Ste12_Rep2 (red), PolII_Rep2 (green) and Input_ACGT (light blue). IGB signal tracks of chromosome 12 between 80,000 and 210,000 are shown for each sample. For (a) and (b), axis and scale normalizations are similar to Figure 2 . (c) Left: Rank-rank comparison of target lists between all pairwise barcoded replicates for Cse4, PolII and Ste12. The horizontal axis shows the fraction of the two lists being compared and the vertical axis shows the fraction of those targets that agree between a given pair of target lists. All comparisons show strong agreement, although the rank lists for Cse4 differ more than PolII or Ste12 for the second half of their length. Right: Rank-rank comparison between barcoded replicates from the same factors (averaged over all pairwise comparisons) compared to rank-rank comparisons for barcoded replicates between different factors: PolII_Rep1 (ACGT) vs. Ste12_Rep1 (TGCT) and Cse4_Rep2 (CATT) vs. Ste12_Rep2 (GTAT).
Figure Legend Snippet: Barcoded adapters perform similarly to standard Illumina adapters and do not crossover to other samples in the same lane . (a) RNA PolII binding profiles from different biological replicates with the same barcode (PolII_Rep1, dark blue; PolII_Rep3, red), with different barcodes (PolII_Rep2, orange) or without barcode (PolII_Rep4, green) strongly overlap. See also Table 3 . Input DNA serves as a reference (light blue). IGB signal tracks of chromosome 5 between 130,000 and 320,000 are shown for each library. A box in the left panel depicts the enlarged section shown in the right panel between positions 298,000 and 309,000 to illustrate the overlap among all PolII signal tracks. (b) Binding profiles from four different libraries pooled and sequenced in the same flowcell lane show very little resemblance. Shown here are the binding profiles for Cse4_Rep2 (dark blue), Ste12_Rep2 (red), PolII_Rep2 (green) and Input_ACGT (light blue). IGB signal tracks of chromosome 12 between 80,000 and 210,000 are shown for each sample. For (a) and (b), axis and scale normalizations are similar to Figure 2 . (c) Left: Rank-rank comparison of target lists between all pairwise barcoded replicates for Cse4, PolII and Ste12. The horizontal axis shows the fraction of the two lists being compared and the vertical axis shows the fraction of those targets that agree between a given pair of target lists. All comparisons show strong agreement, although the rank lists for Cse4 differ more than PolII or Ste12 for the second half of their length. Right: Rank-rank comparison between barcoded replicates from the same factors (averaged over all pairwise comparisons) compared to rank-rank comparisons for barcoded replicates between different factors: PolII_Rep1 (ACGT) vs. Ste12_Rep1 (TGCT) and Cse4_Rep2 (CATT) vs. Ste12_Rep2 (GTAT).

Techniques Used: Binding Assay

Scheme for yeast barcoded ChIP-Seq . (a) Barcoded ChIP-Seq workflow. Ovals depict yeast cells and squares depict proteins. An aliquot of sheared cell lysate is not immunoprecipitated but is otherwise processed normally (green). This DNA, termed input DNA, is a reference sample for ChIP-Seq. Illumina DNA libraries are generated from both ChIP and input DNA samples. In multiplex ChIP-Seq, a barcoded adapter is ligated to an individual DNA sample. The barcode has 3 unique bases followed by a 'T' to anneal with the end-repaired DNA. Four libraries are then pooled together and applied to a single flowcell lane. After sequencing on the Genome Analyzer, reads are separated according to the first four bases and aligned to the yeast genome. Reads are stacked to generate a signal profile and scored against a pool of input DNA to determine significant transcription factor binding sites. (b) The barcode (orange) is located between Illumina adapter sequences (purple) and ChIP or input DNA inserts (black). The sequencing primer (pink) anneals to the adapter sequences and short reads start with the four bases of the barcode (orange) followed by DNA inserts (black). For the sequencing primer and Illumina adapter, oligonucleotide sequences were given by the manufacturer © 2006 Illumina, Inc. All rights reserved.
Figure Legend Snippet: Scheme for yeast barcoded ChIP-Seq . (a) Barcoded ChIP-Seq workflow. Ovals depict yeast cells and squares depict proteins. An aliquot of sheared cell lysate is not immunoprecipitated but is otherwise processed normally (green). This DNA, termed input DNA, is a reference sample for ChIP-Seq. Illumina DNA libraries are generated from both ChIP and input DNA samples. In multiplex ChIP-Seq, a barcoded adapter is ligated to an individual DNA sample. The barcode has 3 unique bases followed by a 'T' to anneal with the end-repaired DNA. Four libraries are then pooled together and applied to a single flowcell lane. After sequencing on the Genome Analyzer, reads are separated according to the first four bases and aligned to the yeast genome. Reads are stacked to generate a signal profile and scored against a pool of input DNA to determine significant transcription factor binding sites. (b) The barcode (orange) is located between Illumina adapter sequences (purple) and ChIP or input DNA inserts (black). The sequencing primer (pink) anneals to the adapter sequences and short reads start with the four bases of the barcode (orange) followed by DNA inserts (black). For the sequencing primer and Illumina adapter, oligonucleotide sequences were given by the manufacturer © 2006 Illumina, Inc. All rights reserved.

Techniques Used: Chromatin Immunoprecipitation, Immunoprecipitation, Generated, Multiplex Assay, Sequencing, Binding Assay

13) Product Images from "Efficient yeast ChIP-Seq using multiplex short-read DNA sequencing"

Article Title: Efficient yeast ChIP-Seq using multiplex short-read DNA sequencing

Journal: BMC Genomics

doi: 10.1186/1471-2164-10-37

PolII signal profiles recapitulate findings from Steinmetz et al . PolII ChIP-Seq signal profiles resemble very closely to those published in Figure 3 of Steinmetz et al [ 54 ]. We obtained consistent binding at the Bap2-Tat1 loci (a) and at the Sed1-Shu2 loci (b). As expected, we did not observe binding at the Flo11 locus (c). For PolII ChIP-Seq experiments, two biological replicates were barcoded with ACGT (PolII_Rep1, dark blue; PolII_Rep2, orange), one was barcoded with TGCT (PolII_Rep3, red) and a fourth replicate had non-barcoded adapters (PolII_Rep4, green). Input DNA serves as a reference (light blue). Axis and scale normalizations are similar to Figure 2 . ORFs above the coordinates axis are on the Watson strand while ORFs below this axis are on the Crick strand.
Figure Legend Snippet: PolII signal profiles recapitulate findings from Steinmetz et al . PolII ChIP-Seq signal profiles resemble very closely to those published in Figure 3 of Steinmetz et al [ 54 ]. We obtained consistent binding at the Bap2-Tat1 loci (a) and at the Sed1-Shu2 loci (b). As expected, we did not observe binding at the Flo11 locus (c). For PolII ChIP-Seq experiments, two biological replicates were barcoded with ACGT (PolII_Rep1, dark blue; PolII_Rep2, orange), one was barcoded with TGCT (PolII_Rep3, red) and a fourth replicate had non-barcoded adapters (PolII_Rep4, green). Input DNA serves as a reference (light blue). Axis and scale normalizations are similar to Figure 2 . ORFs above the coordinates axis are on the Watson strand while ORFs below this axis are on the Crick strand.

Techniques Used: Chromatin Immunoprecipitation, Binding Assay

Comparison of input DNA signal tracks among all four barcoded adapters relative to standard Illumina adapters . An input sample was split in five aliquots. Four were barcoded differentially (top four lanes) and one had non-barcoded, Illumina adapters (fifth lane, labeled 'None'). Barcoded inputs were scored against non-barcoded input. IGB signal tracks of yeast chromosome 16 are shown for each sample, with ORF locations on the x-axis. ORFs are depicted in purple. On the y-axis, a normalized scale represents the number of read counts at a particular location. Each scale is normalized according to the number of mapped reads (Table 10 ). A box in the left panel depicts the enlarged section shown in the right panel for positions between 828,000 and 833,000 to demonstrate the overlap among all signal tracks.
Figure Legend Snippet: Comparison of input DNA signal tracks among all four barcoded adapters relative to standard Illumina adapters . An input sample was split in five aliquots. Four were barcoded differentially (top four lanes) and one had non-barcoded, Illumina adapters (fifth lane, labeled 'None'). Barcoded inputs were scored against non-barcoded input. IGB signal tracks of yeast chromosome 16 are shown for each sample, with ORF locations on the x-axis. ORFs are depicted in purple. On the y-axis, a normalized scale represents the number of read counts at a particular location. Each scale is normalized according to the number of mapped reads (Table 10 ). A box in the left panel depicts the enlarged section shown in the right panel for positions between 828,000 and 833,000 to demonstrate the overlap among all signal tracks.

Techniques Used: Labeling

Barcoded adapters perform similarly to standard Illumina adapters and do not crossover to other samples in the same lane . (a) RNA PolII binding profiles from different biological replicates with the same barcode (PolII_Rep1, dark blue; PolII_Rep3, red), with different barcodes (PolII_Rep2, orange) or without barcode (PolII_Rep4, green) strongly overlap. See also Table 3 . Input DNA serves as a reference (light blue). IGB signal tracks of chromosome 5 between 130,000 and 320,000 are shown for each library. A box in the left panel depicts the enlarged section shown in the right panel between positions 298,000 and 309,000 to illustrate the overlap among all PolII signal tracks. (b) Binding profiles from four different libraries pooled and sequenced in the same flowcell lane show very little resemblance. Shown here are the binding profiles for Cse4_Rep2 (dark blue), Ste12_Rep2 (red), PolII_Rep2 (green) and Input_ACGT (light blue). IGB signal tracks of chromosome 12 between 80,000 and 210,000 are shown for each sample. For (a) and (b), axis and scale normalizations are similar to Figure 2 . (c) Left: Rank-rank comparison of target lists between all pairwise barcoded replicates for Cse4, PolII and Ste12. The horizontal axis shows the fraction of the two lists being compared and the vertical axis shows the fraction of those targets that agree between a given pair of target lists. All comparisons show strong agreement, although the rank lists for Cse4 differ more than PolII or Ste12 for the second half of their length. Right: Rank-rank comparison between barcoded replicates from the same factors (averaged over all pairwise comparisons) compared to rank-rank comparisons for barcoded replicates between different factors: PolII_Rep1 (ACGT) vs. Ste12_Rep1 (TGCT) and Cse4_Rep2 (CATT) vs. Ste12_Rep2 (GTAT).
Figure Legend Snippet: Barcoded adapters perform similarly to standard Illumina adapters and do not crossover to other samples in the same lane . (a) RNA PolII binding profiles from different biological replicates with the same barcode (PolII_Rep1, dark blue; PolII_Rep3, red), with different barcodes (PolII_Rep2, orange) or without barcode (PolII_Rep4, green) strongly overlap. See also Table 3 . Input DNA serves as a reference (light blue). IGB signal tracks of chromosome 5 between 130,000 and 320,000 are shown for each library. A box in the left panel depicts the enlarged section shown in the right panel between positions 298,000 and 309,000 to illustrate the overlap among all PolII signal tracks. (b) Binding profiles from four different libraries pooled and sequenced in the same flowcell lane show very little resemblance. Shown here are the binding profiles for Cse4_Rep2 (dark blue), Ste12_Rep2 (red), PolII_Rep2 (green) and Input_ACGT (light blue). IGB signal tracks of chromosome 12 between 80,000 and 210,000 are shown for each sample. For (a) and (b), axis and scale normalizations are similar to Figure 2 . (c) Left: Rank-rank comparison of target lists between all pairwise barcoded replicates for Cse4, PolII and Ste12. The horizontal axis shows the fraction of the two lists being compared and the vertical axis shows the fraction of those targets that agree between a given pair of target lists. All comparisons show strong agreement, although the rank lists for Cse4 differ more than PolII or Ste12 for the second half of their length. Right: Rank-rank comparison between barcoded replicates from the same factors (averaged over all pairwise comparisons) compared to rank-rank comparisons for barcoded replicates between different factors: PolII_Rep1 (ACGT) vs. Ste12_Rep1 (TGCT) and Cse4_Rep2 (CATT) vs. Ste12_Rep2 (GTAT).

Techniques Used: Binding Assay

Ste12 distribution during pseudohyphal growth is similar across three different biological replicates . Two barcoded replicates (Ste12_Rep2, dark blue; Ste12_Rep1, red) and a non-barcoded replicate (Ste12_Rep3, green) were compared to input DNA (light blue). Ste12 ChIP samples were scored against a pool of input DNA. IGB signal tracks of chromosome 2 between 340,000 and 410,000 are shown for each sample. Axis and scale normalizations are similar to Figure 2 . A box in the left panel containing the TEC1 gene and its surrounding intergenic region was enlarged in panel B and rescaled to emphasize the strong signal at the TEC1 promoter. The same normalization as in Figure 2 was applied. Ste12p and Tec1p act as a dimer during pseudohyphal growth [ 31 ].
Figure Legend Snippet: Ste12 distribution during pseudohyphal growth is similar across three different biological replicates . Two barcoded replicates (Ste12_Rep2, dark blue; Ste12_Rep1, red) and a non-barcoded replicate (Ste12_Rep3, green) were compared to input DNA (light blue). Ste12 ChIP samples were scored against a pool of input DNA. IGB signal tracks of chromosome 2 between 340,000 and 410,000 are shown for each sample. Axis and scale normalizations are similar to Figure 2 . A box in the left panel containing the TEC1 gene and its surrounding intergenic region was enlarged in panel B and rescaled to emphasize the strong signal at the TEC1 promoter. The same normalization as in Figure 2 was applied. Ste12p and Tec1p act as a dimer during pseudohyphal growth [ 31 ].

Techniques Used: Chromatin Immunoprecipitation, Activated Clotting Time Assay

Cse4p is found robustly at centromeres . All biological replicates were strongly and tightly bound to centromeres, as it is depicted here in the case of CEN11 . Two barcoded replicates (Cse4_Rep2, dark blue; Cse4_Rep1, red) and a non-barcoded replicate (Cse4_Rep3, green) were compared to input DNA (light blue). Cse4 ChIP samples were scored against a pool of input DNA. IGB signal tracks of the CEN11 on chromosome 11 are shown for each sample. CEN11 is highlighted in a yellow box. Axis and scale normalizations are similar to Figure 2 .
Figure Legend Snippet: Cse4p is found robustly at centromeres . All biological replicates were strongly and tightly bound to centromeres, as it is depicted here in the case of CEN11 . Two barcoded replicates (Cse4_Rep2, dark blue; Cse4_Rep1, red) and a non-barcoded replicate (Cse4_Rep3, green) were compared to input DNA (light blue). Cse4 ChIP samples were scored against a pool of input DNA. IGB signal tracks of the CEN11 on chromosome 11 are shown for each sample. CEN11 is highlighted in a yellow box. Axis and scale normalizations are similar to Figure 2 .

Techniques Used: Chromatin Immunoprecipitation

Scheme for yeast barcoded ChIP-Seq . (a) Barcoded ChIP-Seq workflow. Ovals depict yeast cells and squares depict proteins. An aliquot of sheared cell lysate is not immunoprecipitated but is otherwise processed normally (green). This DNA, termed input DNA, is a reference sample for ChIP-Seq. Illumina DNA libraries are generated from both ChIP and input DNA samples. In multiplex ChIP-Seq, a barcoded adapter is ligated to an individual DNA sample. The barcode has 3 unique bases followed by a 'T' to anneal with the end-repaired DNA. Four libraries are then pooled together and applied to a single flowcell lane. After sequencing on the Genome Analyzer, reads are separated according to the first four bases and aligned to the yeast genome. Reads are stacked to generate a signal profile and scored against a pool of input DNA to determine significant transcription factor binding sites. (b) The barcode (orange) is located between Illumina adapter sequences (purple) and ChIP or input DNA inserts (black). The sequencing primer (pink) anneals to the adapter sequences and short reads start with the four bases of the barcode (orange) followed by DNA inserts (black). For the sequencing primer and Illumina adapter, oligonucleotide sequences were given by the manufacturer © 2006 Illumina, Inc. All rights reserved.
Figure Legend Snippet: Scheme for yeast barcoded ChIP-Seq . (a) Barcoded ChIP-Seq workflow. Ovals depict yeast cells and squares depict proteins. An aliquot of sheared cell lysate is not immunoprecipitated but is otherwise processed normally (green). This DNA, termed input DNA, is a reference sample for ChIP-Seq. Illumina DNA libraries are generated from both ChIP and input DNA samples. In multiplex ChIP-Seq, a barcoded adapter is ligated to an individual DNA sample. The barcode has 3 unique bases followed by a 'T' to anneal with the end-repaired DNA. Four libraries are then pooled together and applied to a single flowcell lane. After sequencing on the Genome Analyzer, reads are separated according to the first four bases and aligned to the yeast genome. Reads are stacked to generate a signal profile and scored against a pool of input DNA to determine significant transcription factor binding sites. (b) The barcode (orange) is located between Illumina adapter sequences (purple) and ChIP or input DNA inserts (black). The sequencing primer (pink) anneals to the adapter sequences and short reads start with the four bases of the barcode (orange) followed by DNA inserts (black). For the sequencing primer and Illumina adapter, oligonucleotide sequences were given by the manufacturer © 2006 Illumina, Inc. All rights reserved.

Techniques Used: Chromatin Immunoprecipitation, Immunoprecipitation, Generated, Multiplex Assay, Sequencing, Binding Assay

14) Product Images from "PEG3 control on the mammalian MSL complex"

Article Title: PEG3 control on the mammalian MSL complex

Journal: PLoS ONE

doi: 10.1371/journal.pone.0178363

PEG3 binding to the promoter regions of Msl1 and Msl3 . ( A-B ) Two sets of the results derived from ChIP-seq experiments using anti-PEG3 antibody are shown with the 100-kb genomic intervals containing Msl1 (Mmu11) and Msl3 (MmuX). The panels on top are from the WT-MEF cells, whereas the panels on bottom are from the KO-MEF cells. The values on the Y-axis indicate statistical p values, and all four panels have been presented on the same scale with the maximum value being 30. The values on the X-axis indicate the relative genomic positions, and the transcriptional direction of each gene is indicated with an arrow. ( C ) Individual ChIP experiments confirming the binding of PEG3 to the promoter regions of Msl1 and Msl3 . The immunoprecipitated DNA from WT or KO-MEF cells with anti-PEG3 antibody was used as template DNA for PCR amplification. This series of PCR surveys also included the two controls as template DNA: the Input and Negative (Neg). The Neg control was derived from the ChIP experiment without the antibody. ( D ) Quantitative PCR analyses of the immunoprecipitated DNA with anti-PEG3 antibody. The enrichment levels at the promoter regions of Msl1 and Msl3 were measured and compared between the Neg and PEG3-IP samples. The values on the Y-axis indicate the relative enrichment value of each sample to the amount of the Input sample. This series of analyses were performed in triplicates and also repeated more than two independent trials.
Figure Legend Snippet: PEG3 binding to the promoter regions of Msl1 and Msl3 . ( A-B ) Two sets of the results derived from ChIP-seq experiments using anti-PEG3 antibody are shown with the 100-kb genomic intervals containing Msl1 (Mmu11) and Msl3 (MmuX). The panels on top are from the WT-MEF cells, whereas the panels on bottom are from the KO-MEF cells. The values on the Y-axis indicate statistical p values, and all four panels have been presented on the same scale with the maximum value being 30. The values on the X-axis indicate the relative genomic positions, and the transcriptional direction of each gene is indicated with an arrow. ( C ) Individual ChIP experiments confirming the binding of PEG3 to the promoter regions of Msl1 and Msl3 . The immunoprecipitated DNA from WT or KO-MEF cells with anti-PEG3 antibody was used as template DNA for PCR amplification. This series of PCR surveys also included the two controls as template DNA: the Input and Negative (Neg). The Neg control was derived from the ChIP experiment without the antibody. ( D ) Quantitative PCR analyses of the immunoprecipitated DNA with anti-PEG3 antibody. The enrichment levels at the promoter regions of Msl1 and Msl3 were measured and compared between the Neg and PEG3-IP samples. The values on the Y-axis indicate the relative enrichment value of each sample to the amount of the Input sample. This series of analyses were performed in triplicates and also repeated more than two independent trials.

Techniques Used: Binding Assay, Derivative Assay, Chromatin Immunoprecipitation, Immunoprecipitation, Polymerase Chain Reaction, Amplification, Real-time Polymerase Chain Reaction

15) Product Images from "Gene Expression and Chromatin Modifications Associated with Maize Centromeres"

Article Title: Gene Expression and Chromatin Modifications Associated with Maize Centromeres

Journal: G3: Genes|Genomes|Genetics

doi: 10.1534/g3.115.022764

Positioning of CENH3 nucleosomes and MNase cutting patterns associated with CentC repeat. (A) Well-positioned CENH3 nucleosomes in Cen2 , as indicated by CENH3 ChIP-seq reads. Top panel, a region containing an array of 20 CentC monomers marked by green bars. Most peaks were phased with individual CentC monomers. Bottom panel: a region containing Ty3 / gypsy LTR retrotransposon sequences. Most CENH3 nucleosomes were well positioned but were not phased. (B) MNase cutting pattern on a consensus CentC repeat monomer. Vertical bars represent the frequency of cutting at each position on the CentC monomer. The y -axis shows the number of cuts in ChIPed DNA and naked DNA datasets. The x -axis shows each site of the consensus CentC monomer (156 bp). Black dashed lines mark the top 20% cutting frequency. (C) Phasogram of CENH3 nucleosomes in Cen2 . Total Cen2 : all fragments mapped to Cen2 were used for the phasogram; CentC : only fragments mapped to CentC arrays were calculated for the phasogram. The y -axes is the frequency of distance between the midpoints of two fragments.
Figure Legend Snippet: Positioning of CENH3 nucleosomes and MNase cutting patterns associated with CentC repeat. (A) Well-positioned CENH3 nucleosomes in Cen2 , as indicated by CENH3 ChIP-seq reads. Top panel, a region containing an array of 20 CentC monomers marked by green bars. Most peaks were phased with individual CentC monomers. Bottom panel: a region containing Ty3 / gypsy LTR retrotransposon sequences. Most CENH3 nucleosomes were well positioned but were not phased. (B) MNase cutting pattern on a consensus CentC repeat monomer. Vertical bars represent the frequency of cutting at each position on the CentC monomer. The y -axis shows the number of cuts in ChIPed DNA and naked DNA datasets. The x -axis shows each site of the consensus CentC monomer (156 bp). Black dashed lines mark the top 20% cutting frequency. (C) Phasogram of CENH3 nucleosomes in Cen2 . Total Cen2 : all fragments mapped to Cen2 were used for the phasogram; CentC : only fragments mapped to CentC arrays were calculated for the phasogram. The y -axes is the frequency of distance between the midpoints of two fragments.

Techniques Used: Chromatin Immunoprecipitation

Chromatin immunoprecipitation (ChIP) and sequencing of maize centromeres. (A) Agarose gel electrophoresis of DNAs isolated from maize chromatin digested by 0.2 U and 5 U micrococcal nuclease (MNase), respectively. Libraries were developed from both ChIPed and input DNA samples of both chromatin samples. (B) Distribution of fragment lengths from 0.2 U and 5 U ChIPed and input sequencing libraries. The x -axis represents the length of fragments (bp). The y -axis represents the percentage of reads in specific length.
Figure Legend Snippet: Chromatin immunoprecipitation (ChIP) and sequencing of maize centromeres. (A) Agarose gel electrophoresis of DNAs isolated from maize chromatin digested by 0.2 U and 5 U micrococcal nuclease (MNase), respectively. Libraries were developed from both ChIPed and input DNA samples of both chromatin samples. (B) Distribution of fragment lengths from 0.2 U and 5 U ChIPed and input sequencing libraries. The x -axis represents the length of fragments (bp). The y -axis represents the percentage of reads in specific length.

Techniques Used: Chromatin Immunoprecipitation, Sequencing, Agarose Gel Electrophoresis, Isolation

16) Product Images from "A Comprehensive Profile of ChIP-Seq-Based STAT1 Target Genes Suggests the Complexity of STAT1-Mediated Gene Regulatory Mechanisms"

Article Title: A Comprehensive Profile of ChIP-Seq-Based STAT1 Target Genes Suggests the Complexity of STAT1-Mediated Gene Regulatory Mechanisms

Journal: Gene Regulation and Systems Biology

doi: 10.4137/GRSB.S11433

FastQC analysis of ChIP-Seq data. FASTQ format files are derived from short read NGS data of STAT1-ChIP-treated DNA (Panel A ) and input DNA (Panel B ). Notes: They were imported into the FastQC program. The per base sequence quality score is shown with the median (red line), the mean (blue line), and the interquatile range (yellow box).
Figure Legend Snippet: FastQC analysis of ChIP-Seq data. FASTQ format files are derived from short read NGS data of STAT1-ChIP-treated DNA (Panel A ) and input DNA (Panel B ). Notes: They were imported into the FastQC program. The per base sequence quality score is shown with the median (red line), the mean (blue line), and the interquatile range (yellow box).

Techniques Used: Chromatin Immunoprecipitation, Derivative Assay, Next-Generation Sequencing, Sequencing

17) Product Images from "T-bet and GATA3 orchestrate Th1 and Th2 differentiation through lineage-specific targeting of distal regulatory elements"

Article Title: T-bet and GATA3 orchestrate Th1 and Th2 differentiation through lineage-specific targeting of distal regulatory elements

Journal: Nature Communications

doi: 10.1038/ncomms2260

Markers of enhancer and insulator elements at distal T-bet and GATA3 binding sites. ( a ) Average sequence conservation between 28 sequenced vertebrate genomes ( y axis, phastCons score) plotted against the genomic distance from a T-bet (green, left) or GATA3 (blue, right) binding site ( x axis). ( b ) Average number of sequence reads measuring the location of DHS sites ( y axis) plotted against the genomic distance from T-bet and GATA3 binding sites as before. ( c ) Average number of sequence reads from H3K4me1 ChIP DNA from resting T cells. ( d ) Average number of sequence reads from CTCF ChIP DNA from resting T cells. ( e ) Luciferase activity (firefly/renilla relative to Ifng promoter, mean and s.d., three independent transfections, performed in duplicate) in human Th0 cells transfected with Ifng promoter-driven luciferase reporter constructs with or without the named distal elements inserted upstream. *Significant increase in luciferase activity at P
Figure Legend Snippet: Markers of enhancer and insulator elements at distal T-bet and GATA3 binding sites. ( a ) Average sequence conservation between 28 sequenced vertebrate genomes ( y axis, phastCons score) plotted against the genomic distance from a T-bet (green, left) or GATA3 (blue, right) binding site ( x axis). ( b ) Average number of sequence reads measuring the location of DHS sites ( y axis) plotted against the genomic distance from T-bet and GATA3 binding sites as before. ( c ) Average number of sequence reads from H3K4me1 ChIP DNA from resting T cells. ( d ) Average number of sequence reads from CTCF ChIP DNA from resting T cells. ( e ) Luciferase activity (firefly/renilla relative to Ifng promoter, mean and s.d., three independent transfections, performed in duplicate) in human Th0 cells transfected with Ifng promoter-driven luciferase reporter constructs with or without the named distal elements inserted upstream. *Significant increase in luciferase activity at P

Techniques Used: Binding Assay, Sequencing, Chromatin Immunoprecipitation, Luciferase, Activity Assay, Transfection, Construct

T-bet and GATA3 binding sites at human Th1 and Th2 cytokine loci. ( a ) T-bet and GATA3 binding at the IFNG locus (region shown chr12:66,650,000–66,950,000 (hg18) in reverse orientation). The number of sequencing reads from T-bet and GATA3 ChIP-enriched DNA are plotted per million background-subtracted total reads and aligned with the human genome. DHS sites in Th1 and Th2 cells are marked by dashes and sites of H3K4me1 and CTCF occupancy in resting T cells are shown below as reads per million. The positions of orthologous murine regulatory elements are indicated by the bars underneath and labelled with the approximate distance of the associated T-bet peak to the IFNG TSS. Gene structures are marked at the bottom of the figure and the start and direction of transcription with arrows. ( b ) As a , except for the IL4/5/13 locus (region shown chr5:131,880,000–132,090,000). Murine regulatory elements are marked; LCR, locus control region; CGRE, CG-rich element; CNS, conserved non-coding sequence; SIL, silencer.
Figure Legend Snippet: T-bet and GATA3 binding sites at human Th1 and Th2 cytokine loci. ( a ) T-bet and GATA3 binding at the IFNG locus (region shown chr12:66,650,000–66,950,000 (hg18) in reverse orientation). The number of sequencing reads from T-bet and GATA3 ChIP-enriched DNA are plotted per million background-subtracted total reads and aligned with the human genome. DHS sites in Th1 and Th2 cells are marked by dashes and sites of H3K4me1 and CTCF occupancy in resting T cells are shown below as reads per million. The positions of orthologous murine regulatory elements are indicated by the bars underneath and labelled with the approximate distance of the associated T-bet peak to the IFNG TSS. Gene structures are marked at the bottom of the figure and the start and direction of transcription with arrows. ( b ) As a , except for the IL4/5/13 locus (region shown chr5:131,880,000–132,090,000). Murine regulatory elements are marked; LCR, locus control region; CGRE, CG-rich element; CNS, conserved non-coding sequence; SIL, silencer.

Techniques Used: Binding Assay, Sequencing, Chromatin Immunoprecipitation

T-bet co-expression induces GATA3 occupancy at T-bet binding sites. ( a ) Enrichment of Il4 CNS2 DNA (mean and s.d., n =3) by ChIP with anti-FLAG or anti-HA antibodies relative to input DNA measured by quantitative PCR. ChIPs were performed in four stable EL4 cell lines expressing FLAG-tagged T-bet, HA-tagged Gata3, neither protein or both proteins together. Data are normalized to a control region within Dleu2 that lacks T-bet and GATA3 binding sites. ( b ) As a ., except for Ifng CNS-6. ( c ) As a , except for the murine ortholog of the Tbx21 −11-kb element. ( d ) Model. T-bet and GATA3 act through distal elements to control expression of immune regulator genes. The alternate distribution of GATA3 from Th2-specific GATA sites to a set of Th1-specific T-bet binding sites is associated with a switch from Th2 to Th1-specific expression.
Figure Legend Snippet: T-bet co-expression induces GATA3 occupancy at T-bet binding sites. ( a ) Enrichment of Il4 CNS2 DNA (mean and s.d., n =3) by ChIP with anti-FLAG or anti-HA antibodies relative to input DNA measured by quantitative PCR. ChIPs were performed in four stable EL4 cell lines expressing FLAG-tagged T-bet, HA-tagged Gata3, neither protein or both proteins together. Data are normalized to a control region within Dleu2 that lacks T-bet and GATA3 binding sites. ( b ) As a ., except for Ifng CNS-6. ( c ) As a , except for the murine ortholog of the Tbx21 −11-kb element. ( d ) Model. T-bet and GATA3 act through distal elements to control expression of immune regulator genes. The alternate distribution of GATA3 from Th2-specific GATA sites to a set of Th1-specific T-bet binding sites is associated with a switch from Th2 to Th1-specific expression.

Techniques Used: Expressing, Binding Assay, Chromatin Immunoprecipitation, Real-time Polymerase Chain Reaction, Activated Clotting Time Assay

18) Product Images from "Deep sequencing reveals distinct patterns of DNA methylation in prostate cancer"

Article Title: Deep sequencing reveals distinct patterns of DNA methylation in prostate cancer

Journal: Genome Research

doi: 10.1101/gr.119347.110

Mutually exclusive patterns of promoter DNA methylation and histone H3K4me3 marks in LNCaP cells. Integration of M-NGS DNA methylation data with H3K4me3 ChIP-seq data indicates that DNA methylation and H3K4me3 may be present on the same gene promoter
Figure Legend Snippet: Mutually exclusive patterns of promoter DNA methylation and histone H3K4me3 marks in LNCaP cells. Integration of M-NGS DNA methylation data with H3K4me3 ChIP-seq data indicates that DNA methylation and H3K4me3 may be present on the same gene promoter

Techniques Used: DNA Methylation Assay, Next-Generation Sequencing, Chromatin Immunoprecipitation

19) Product Images from "Shallow whole genome sequencing for robust copy number profiling of formalin-fixed paraffin-embedded breast cancers"

Article Title: Shallow whole genome sequencing for robust copy number profiling of formalin-fixed paraffin-embedded breast cancers

Journal: Experimental and Molecular Pathology

doi: 10.1016/j.yexmp.2018.03.006

Features of the sequencing libraries. Boxplots showing different features of input DNA and library yield relative to the different library qualities. A. Input DNA. B. Quality of input DNA inferred from ΔCt. C. Fragment size of libraries. D. Library yield. VG = very good, G = good, I = intermediate, P = poor, F = fail.
Figure Legend Snippet: Features of the sequencing libraries. Boxplots showing different features of input DNA and library yield relative to the different library qualities. A. Input DNA. B. Quality of input DNA inferred from ΔCt. C. Fragment size of libraries. D. Library yield. VG = very good, G = good, I = intermediate, P = poor, F = fail.

Techniques Used: Sequencing

Features of input DNA and libraries generated from blocks less and more than five years. Dot plots represent the range (minimum-maximum) of observed values for each of the following categories and the red dot represents the median. A. The quality of input DNA inferred by ΔCt. B. Fragment sizes of the libraries in base pair. C. The library yield in nanomoles.
Figure Legend Snippet: Features of input DNA and libraries generated from blocks less and more than five years. Dot plots represent the range (minimum-maximum) of observed values for each of the following categories and the red dot represents the median. A. The quality of input DNA inferred by ΔCt. B. Fragment sizes of the libraries in base pair. C. The library yield in nanomoles.

Techniques Used: Generated

20) Product Images from "PEG3 binds to H19-ICR as a transcriptional repressor"

Article Title: PEG3 binds to H19-ICR as a transcriptional repressor

Journal: Epigenetics

doi: 10.1080/15592294.2016.1255385

PEG3 binding to the H19-ICR. Individual ChIP experiments were performed to confirm the in vivo binding of PEG to the H19-ICR using 2 sets of chromatins prepared from MEF cells and neonatal brains (A). Each set of chromatin was also derived from 2 different samples, representing WT and KO (+/-p) cells. Since Peg3 is expressed mainly from the paternal allele, the heterozygotes with the paternal transmission are considered to be null. Three individual DNA were derived from each ChIP experiment, and used as templates for PCR survey: Input, Negative control (Neg), and the immunoprecipitated DNA with anti-PEG3 antibody (PEG3 IP). PCR-based surveys tested 3 individual regions: Pgm2l1 as a positive control that has been known to be bound by PEG3, H19 ICR to test the 420-bp-long peak region, and H19 Peak to test the 152-bp-long narrower peak region of the H19-ICR. (B) Allele test of PEG3 binding. Individual ChIP experiments were repeated with the chromatin isolated from the 11.5-dpc F1 hybrid embryos that had been prepared through the crossing between male Spretus and female C57BL/6J (B6). A restriction enzyme digestion ( Acu I) showed 2 alleles in the Input as well as the immunoprecipitated DNA, but with different ratios between the 2 alleles, which are shown underneath the gel images. (C) Imprinting test of H19 expression. Total RNA was isolated from 2 sets of the F1 hybrid samples and 2 sets of hybrid MEF cell samples that had been prepared through the crossing between male B6 and female PWD/PhJ. A restriction enzyme digestion ( Bcl I) showed the 2 parental alleles (lane 1–2), and also the maternal-specific expression in neonatal brains (lane 3–4) and in MEF cells (lane 5–6).
Figure Legend Snippet: PEG3 binding to the H19-ICR. Individual ChIP experiments were performed to confirm the in vivo binding of PEG to the H19-ICR using 2 sets of chromatins prepared from MEF cells and neonatal brains (A). Each set of chromatin was also derived from 2 different samples, representing WT and KO (+/-p) cells. Since Peg3 is expressed mainly from the paternal allele, the heterozygotes with the paternal transmission are considered to be null. Three individual DNA were derived from each ChIP experiment, and used as templates for PCR survey: Input, Negative control (Neg), and the immunoprecipitated DNA with anti-PEG3 antibody (PEG3 IP). PCR-based surveys tested 3 individual regions: Pgm2l1 as a positive control that has been known to be bound by PEG3, H19 ICR to test the 420-bp-long peak region, and H19 Peak to test the 152-bp-long narrower peak region of the H19-ICR. (B) Allele test of PEG3 binding. Individual ChIP experiments were repeated with the chromatin isolated from the 11.5-dpc F1 hybrid embryos that had been prepared through the crossing between male Spretus and female C57BL/6J (B6). A restriction enzyme digestion ( Acu I) showed 2 alleles in the Input as well as the immunoprecipitated DNA, but with different ratios between the 2 alleles, which are shown underneath the gel images. (C) Imprinting test of H19 expression. Total RNA was isolated from 2 sets of the F1 hybrid samples and 2 sets of hybrid MEF cell samples that had been prepared through the crossing between male B6 and female PWD/PhJ. A restriction enzyme digestion ( Bcl I) showed the 2 parental alleles (lane 1–2), and also the maternal-specific expression in neonatal brains (lane 3–4) and in MEF cells (lane 5–6).

Techniques Used: Binding Assay, Chromatin Immunoprecipitation, In Vivo, Derivative Assay, Transmission Assay, Polymerase Chain Reaction, Negative Control, Immunoprecipitation, Positive Control, Isolation, Expressing

21) Product Images from "Global Analysis of Transcription Factor-Binding Sites in Yeast Using ChIP-Seq"

Article Title: Global Analysis of Transcription Factor-Binding Sites in Yeast Using ChIP-Seq

Journal: Methods in molecular biology (Clifton, N.J.)

doi: 10.1007/978-1-4939-1363-3_15

Overview of the ChIP-Seq procedure in budding yeast, focusing on a multiplex high-throughput DNA sequencing approach on the Illumina platform
Figure Legend Snippet: Overview of the ChIP-Seq procedure in budding yeast, focusing on a multiplex high-throughput DNA sequencing approach on the Illumina platform

Techniques Used: Chromatin Immunoprecipitation, Multiplex Assay, High Throughput Screening Assay, DNA Sequencing

22) Product Images from "A streamlined method for analysing genome-wide DNA methylation patterns from low amounts of FFPE DNA"

Article Title: A streamlined method for analysing genome-wide DNA methylation patterns from low amounts of FFPE DNA

Journal: BMC Medical Genomics

doi: 10.1186/s12920-017-0290-1

MLH1 PCR to test efficiency of bisulfite conversion on FFPE derived DNA. a ) MLH1 PCR of FFPE-derived bisulfite-treated DNA. Lane 1: 1Kb + ladder, Lane 2: 200 ng input DNA, Lane 3: 500 ng input DNA, Lane 4: Zymo methylated control DNA, Lane 5: unconverted genomic DNA, Lane 6: PCR negative (water). 2% agarose, run for 25 mins at 100 V. b ) MLH1 PCR of RRBS libraries prepared from different amounts of FFPE-derived DNA. FFPE DNA was digested with MspI enzyme, A-tailed, end repaired and ligated to Illumina adaptors, and bisulfite converted. Then PCR was performed with MLH1 primers. Lane 1: 1Kb + ladder, Lane 2: 50 ng input DNA, Lane 3: 100 ng input DNA, Lane 4: 500 ng input DNA, Lane 5: Zymo methylated control DNA, Lane 6: PCR negative (water). 2% agarose, run for 25 mins at 100 V
Figure Legend Snippet: MLH1 PCR to test efficiency of bisulfite conversion on FFPE derived DNA. a ) MLH1 PCR of FFPE-derived bisulfite-treated DNA. Lane 1: 1Kb + ladder, Lane 2: 200 ng input DNA, Lane 3: 500 ng input DNA, Lane 4: Zymo methylated control DNA, Lane 5: unconverted genomic DNA, Lane 6: PCR negative (water). 2% agarose, run for 25 mins at 100 V. b ) MLH1 PCR of RRBS libraries prepared from different amounts of FFPE-derived DNA. FFPE DNA was digested with MspI enzyme, A-tailed, end repaired and ligated to Illumina adaptors, and bisulfite converted. Then PCR was performed with MLH1 primers. Lane 1: 1Kb + ladder, Lane 2: 50 ng input DNA, Lane 3: 100 ng input DNA, Lane 4: 500 ng input DNA, Lane 5: Zymo methylated control DNA, Lane 6: PCR negative (water). 2% agarose, run for 25 mins at 100 V

Techniques Used: Polymerase Chain Reaction, Formalin-fixed Paraffin-Embedded, Derivative Assay, Methylation

Bioanalyser images demonstrating quality of two FFPE RRBS libraries. Each of the RRBS libraries (FFPE1 and FFPE2) was run on an Agilent 2100 Bioanalyzer using the high sensitivity DNA kit. The electropherogram displays a plot of fragment size (bp) versus fluorescence intensity. Peaks at 35 bp and 10,380 bp represent lower and upper markers. The 160–340 bp peaks represent the RRBS library
Figure Legend Snippet: Bioanalyser images demonstrating quality of two FFPE RRBS libraries. Each of the RRBS libraries (FFPE1 and FFPE2) was run on an Agilent 2100 Bioanalyzer using the high sensitivity DNA kit. The electropherogram displays a plot of fragment size (bp) versus fluorescence intensity. Peaks at 35 bp and 10,380 bp represent lower and upper markers. The 160–340 bp peaks represent the RRBS library

Techniques Used: Formalin-fixed Paraffin-Embedded, Fluorescence

23) Product Images from "Reproducibility and reliability of SNP analysis using human cellular DNA at or near nanogram levels"

Article Title: Reproducibility and reliability of SNP analysis using human cellular DNA at or near nanogram levels

Journal: BMC Research Notes

doi: 10.1186/1756-0500-6-515

Detection of complete homozygosity of the short arm including a small deletion in the chromosome 9. The B allele frequency (BAF) and Log R ratio (LRR) plots of SNP calls on chromosome 9 (idiogram displayed on the bottom of the diagram) using various quantities of DNA for the array are shown. The BAF in the short arm are 1 or 0, except the small region of deletion, indicating homozygosity of the entire short arm. The deleted region is indicated with an arrow on the top of the figure. The LRR is clearly absent from 0, which would indicate two copies of the chromosome are present. It is apparent that as the DNA quantity decreases, the level of noise and scattering increases in both plots. However, the region of deletion is clearly identifiable in all arrays.
Figure Legend Snippet: Detection of complete homozygosity of the short arm including a small deletion in the chromosome 9. The B allele frequency (BAF) and Log R ratio (LRR) plots of SNP calls on chromosome 9 (idiogram displayed on the bottom of the diagram) using various quantities of DNA for the array are shown. The BAF in the short arm are 1 or 0, except the small region of deletion, indicating homozygosity of the entire short arm. The deleted region is indicated with an arrow on the top of the figure. The LRR is clearly absent from 0, which would indicate two copies of the chromosome are present. It is apparent that as the DNA quantity decreases, the level of noise and scattering increases in both plots. However, the region of deletion is clearly identifiable in all arrays.

Techniques Used:

24) Product Images from "Shallow whole genome sequencing for robust copy number profiling of formalin-fixed paraffin-embedded breast cancers"

Article Title: Shallow whole genome sequencing for robust copy number profiling of formalin-fixed paraffin-embedded breast cancers

Journal: Experimental and Molecular Pathology

doi: 10.1016/j.yexmp.2018.03.006

Measured standard deviations from the QDNASEQ copy number plots and associations with the quality of sequencing libraries. A. Bar charts showing proportion of samples with different input DNA quality (based on ΔCt) in each sequencing quality group. B. Bar charts showing proportion of samples from FFPE blocks of different fragment sizes in each sequencing quality group. C. Bar charts showing proportion of samples with different amount of input DNA in each sequencing quality group. VG = very good, G = good, I = intermediate, P = poor, F = fail.
Figure Legend Snippet: Measured standard deviations from the QDNASEQ copy number plots and associations with the quality of sequencing libraries. A. Bar charts showing proportion of samples with different input DNA quality (based on ΔCt) in each sequencing quality group. B. Bar charts showing proportion of samples from FFPE blocks of different fragment sizes in each sequencing quality group. C. Bar charts showing proportion of samples with different amount of input DNA in each sequencing quality group. VG = very good, G = good, I = intermediate, P = poor, F = fail.

Techniques Used: Sequencing, Formalin-fixed Paraffin-Embedded

25) Product Images from "Efficient yeast ChIP-Seq using multiplex short-read DNA sequencing"

Article Title: Efficient yeast ChIP-Seq using multiplex short-read DNA sequencing

Journal: BMC Genomics

doi: 10.1186/1471-2164-10-37

PolII signal profiles recapitulate findings from Steinmetz et al . PolII ChIP-Seq signal profiles resemble very closely to those published in Figure 3 of Steinmetz et al [ 54 ]. We obtained consistent binding at the Bap2-Tat1 loci (a) and at the Sed1-Shu2 loci (b). As expected, we did not observe binding at the Flo11 locus (c). For PolII ChIP-Seq experiments, two biological replicates were barcoded with ACGT (PolII_Rep1, dark blue; PolII_Rep2, orange), one was barcoded with TGCT (PolII_Rep3, red) and a fourth replicate had non-barcoded adapters (PolII_Rep4, green). Input DNA serves as a reference (light blue). Axis and scale normalizations are similar to Figure 2 . ORFs above the coordinates axis are on the Watson strand while ORFs below this axis are on the Crick strand.
Figure Legend Snippet: PolII signal profiles recapitulate findings from Steinmetz et al . PolII ChIP-Seq signal profiles resemble very closely to those published in Figure 3 of Steinmetz et al [ 54 ]. We obtained consistent binding at the Bap2-Tat1 loci (a) and at the Sed1-Shu2 loci (b). As expected, we did not observe binding at the Flo11 locus (c). For PolII ChIP-Seq experiments, two biological replicates were barcoded with ACGT (PolII_Rep1, dark blue; PolII_Rep2, orange), one was barcoded with TGCT (PolII_Rep3, red) and a fourth replicate had non-barcoded adapters (PolII_Rep4, green). Input DNA serves as a reference (light blue). Axis and scale normalizations are similar to Figure 2 . ORFs above the coordinates axis are on the Watson strand while ORFs below this axis are on the Crick strand.

Techniques Used: Chromatin Immunoprecipitation, Binding Assay

Comparison of input DNA signal tracks among all four barcoded adapters relative to standard Illumina adapters . An input sample was split in five aliquots. Four were barcoded differentially (top four lanes) and one had non-barcoded, Illumina adapters (fifth lane, labeled 'None'). Barcoded inputs were scored against non-barcoded input. IGB signal tracks of yeast chromosome 16 are shown for each sample, with ORF locations on the x-axis. ORFs are depicted in purple. On the y-axis, a normalized scale represents the number of read counts at a particular location. Each scale is normalized according to the number of mapped reads (Table 10 ). A box in the left panel depicts the enlarged section shown in the right panel for positions between 828,000 and 833,000 to demonstrate the overlap among all signal tracks.
Figure Legend Snippet: Comparison of input DNA signal tracks among all four barcoded adapters relative to standard Illumina adapters . An input sample was split in five aliquots. Four were barcoded differentially (top four lanes) and one had non-barcoded, Illumina adapters (fifth lane, labeled 'None'). Barcoded inputs were scored against non-barcoded input. IGB signal tracks of yeast chromosome 16 are shown for each sample, with ORF locations on the x-axis. ORFs are depicted in purple. On the y-axis, a normalized scale represents the number of read counts at a particular location. Each scale is normalized according to the number of mapped reads (Table 10 ). A box in the left panel depicts the enlarged section shown in the right panel for positions between 828,000 and 833,000 to demonstrate the overlap among all signal tracks.

Techniques Used: Labeling

Barcoded adapters perform similarly to standard Illumina adapters and do not crossover to other samples in the same lane . (a) RNA PolII binding profiles from different biological replicates with the same barcode (PolII_Rep1, dark blue; PolII_Rep3, red), with different barcodes (PolII_Rep2, orange) or without barcode (PolII_Rep4, green) strongly overlap. See also Table 3 . Input DNA serves as a reference (light blue). IGB signal tracks of chromosome 5 between 130,000 and 320,000 are shown for each library. A box in the left panel depicts the enlarged section shown in the right panel between positions 298,000 and 309,000 to illustrate the overlap among all PolII signal tracks. (b) Binding profiles from four different libraries pooled and sequenced in the same flowcell lane show very little resemblance. Shown here are the binding profiles for Cse4_Rep2 (dark blue), Ste12_Rep2 (red), PolII_Rep2 (green) and Input_ACGT (light blue). IGB signal tracks of chromosome 12 between 80,000 and 210,000 are shown for each sample. For (a) and (b), axis and scale normalizations are similar to Figure 2 . (c) Left: Rank-rank comparison of target lists between all pairwise barcoded replicates for Cse4, PolII and Ste12. The horizontal axis shows the fraction of the two lists being compared and the vertical axis shows the fraction of those targets that agree between a given pair of target lists. All comparisons show strong agreement, although the rank lists for Cse4 differ more than PolII or Ste12 for the second half of their length. Right: Rank-rank comparison between barcoded replicates from the same factors (averaged over all pairwise comparisons) compared to rank-rank comparisons for barcoded replicates between different factors: PolII_Rep1 (ACGT) vs. Ste12_Rep1 (TGCT) and Cse4_Rep2 (CATT) vs. Ste12_Rep2 (GTAT).
Figure Legend Snippet: Barcoded adapters perform similarly to standard Illumina adapters and do not crossover to other samples in the same lane . (a) RNA PolII binding profiles from different biological replicates with the same barcode (PolII_Rep1, dark blue; PolII_Rep3, red), with different barcodes (PolII_Rep2, orange) or without barcode (PolII_Rep4, green) strongly overlap. See also Table 3 . Input DNA serves as a reference (light blue). IGB signal tracks of chromosome 5 between 130,000 and 320,000 are shown for each library. A box in the left panel depicts the enlarged section shown in the right panel between positions 298,000 and 309,000 to illustrate the overlap among all PolII signal tracks. (b) Binding profiles from four different libraries pooled and sequenced in the same flowcell lane show very little resemblance. Shown here are the binding profiles for Cse4_Rep2 (dark blue), Ste12_Rep2 (red), PolII_Rep2 (green) and Input_ACGT (light blue). IGB signal tracks of chromosome 12 between 80,000 and 210,000 are shown for each sample. For (a) and (b), axis and scale normalizations are similar to Figure 2 . (c) Left: Rank-rank comparison of target lists between all pairwise barcoded replicates for Cse4, PolII and Ste12. The horizontal axis shows the fraction of the two lists being compared and the vertical axis shows the fraction of those targets that agree between a given pair of target lists. All comparisons show strong agreement, although the rank lists for Cse4 differ more than PolII or Ste12 for the second half of their length. Right: Rank-rank comparison between barcoded replicates from the same factors (averaged over all pairwise comparisons) compared to rank-rank comparisons for barcoded replicates between different factors: PolII_Rep1 (ACGT) vs. Ste12_Rep1 (TGCT) and Cse4_Rep2 (CATT) vs. Ste12_Rep2 (GTAT).

Techniques Used: Binding Assay

Ste12 distribution during pseudohyphal growth is similar across three different biological replicates . Two barcoded replicates (Ste12_Rep2, dark blue; Ste12_Rep1, red) and a non-barcoded replicate (Ste12_Rep3, green) were compared to input DNA (light blue). Ste12 ChIP samples were scored against a pool of input DNA. IGB signal tracks of chromosome 2 between 340,000 and 410,000 are shown for each sample. Axis and scale normalizations are similar to Figure 2 . A box in the left panel containing the TEC1 gene and its surrounding intergenic region was enlarged in panel B and rescaled to emphasize the strong signal at the TEC1 promoter. The same normalization as in Figure 2 was applied. Ste12p and Tec1p act as a dimer during pseudohyphal growth [ 31 ].
Figure Legend Snippet: Ste12 distribution during pseudohyphal growth is similar across three different biological replicates . Two barcoded replicates (Ste12_Rep2, dark blue; Ste12_Rep1, red) and a non-barcoded replicate (Ste12_Rep3, green) were compared to input DNA (light blue). Ste12 ChIP samples were scored against a pool of input DNA. IGB signal tracks of chromosome 2 between 340,000 and 410,000 are shown for each sample. Axis and scale normalizations are similar to Figure 2 . A box in the left panel containing the TEC1 gene and its surrounding intergenic region was enlarged in panel B and rescaled to emphasize the strong signal at the TEC1 promoter. The same normalization as in Figure 2 was applied. Ste12p and Tec1p act as a dimer during pseudohyphal growth [ 31 ].

Techniques Used: Chromatin Immunoprecipitation, Activated Clotting Time Assay

Cse4p is found robustly at centromeres . All biological replicates were strongly and tightly bound to centromeres, as it is depicted here in the case of CEN11 . Two barcoded replicates (Cse4_Rep2, dark blue; Cse4_Rep1, red) and a non-barcoded replicate (Cse4_Rep3, green) were compared to input DNA (light blue). Cse4 ChIP samples were scored against a pool of input DNA. IGB signal tracks of the CEN11 on chromosome 11 are shown for each sample. CEN11 is highlighted in a yellow box. Axis and scale normalizations are similar to Figure 2 .
Figure Legend Snippet: Cse4p is found robustly at centromeres . All biological replicates were strongly and tightly bound to centromeres, as it is depicted here in the case of CEN11 . Two barcoded replicates (Cse4_Rep2, dark blue; Cse4_Rep1, red) and a non-barcoded replicate (Cse4_Rep3, green) were compared to input DNA (light blue). Cse4 ChIP samples were scored against a pool of input DNA. IGB signal tracks of the CEN11 on chromosome 11 are shown for each sample. CEN11 is highlighted in a yellow box. Axis and scale normalizations are similar to Figure 2 .

Techniques Used: Chromatin Immunoprecipitation

Scheme for yeast barcoded ChIP-Seq . (a) Barcoded ChIP-Seq workflow. Ovals depict yeast cells and squares depict proteins. An aliquot of sheared cell lysate is not immunoprecipitated but is otherwise processed normally (green). This DNA, termed input DNA, is a reference sample for ChIP-Seq. Illumina DNA libraries are generated from both ChIP and input DNA samples. In multiplex ChIP-Seq, a barcoded adapter is ligated to an individual DNA sample. The barcode has 3 unique bases followed by a 'T' to anneal with the end-repaired DNA. Four libraries are then pooled together and applied to a single flowcell lane. After sequencing on the Genome Analyzer, reads are separated according to the first four bases and aligned to the yeast genome. Reads are stacked to generate a signal profile and scored against a pool of input DNA to determine significant transcription factor binding sites. (b) The barcode (orange) is located between Illumina adapter sequences (purple) and ChIP or input DNA inserts (black). The sequencing primer (pink) anneals to the adapter sequences and short reads start with the four bases of the barcode (orange) followed by DNA inserts (black). For the sequencing primer and Illumina adapter, oligonucleotide sequences were given by the manufacturer © 2006 Illumina, Inc. All rights reserved.
Figure Legend Snippet: Scheme for yeast barcoded ChIP-Seq . (a) Barcoded ChIP-Seq workflow. Ovals depict yeast cells and squares depict proteins. An aliquot of sheared cell lysate is not immunoprecipitated but is otherwise processed normally (green). This DNA, termed input DNA, is a reference sample for ChIP-Seq. Illumina DNA libraries are generated from both ChIP and input DNA samples. In multiplex ChIP-Seq, a barcoded adapter is ligated to an individual DNA sample. The barcode has 3 unique bases followed by a 'T' to anneal with the end-repaired DNA. Four libraries are then pooled together and applied to a single flowcell lane. After sequencing on the Genome Analyzer, reads are separated according to the first four bases and aligned to the yeast genome. Reads are stacked to generate a signal profile and scored against a pool of input DNA to determine significant transcription factor binding sites. (b) The barcode (orange) is located between Illumina adapter sequences (purple) and ChIP or input DNA inserts (black). The sequencing primer (pink) anneals to the adapter sequences and short reads start with the four bases of the barcode (orange) followed by DNA inserts (black). For the sequencing primer and Illumina adapter, oligonucleotide sequences were given by the manufacturer © 2006 Illumina, Inc. All rights reserved.

Techniques Used: Chromatin Immunoprecipitation, Immunoprecipitation, Generated, Multiplex Assay, Sequencing, Binding Assay

26) Product Images from "Haplotyping germline and cancer genomes using high-throughput linked-read sequencing"

Article Title: Haplotyping germline and cancer genomes using high-throughput linked-read sequencing

Journal: Nature biotechnology

doi: 10.1038/nbt.3432

Overview of the technology for generating linked-reads (a) Gel beads loaded with primers and barcoded oligonucleotides are first mixed with DNA and enzyme mixture, and subsequently mixed with oil-surfactant solution at a microfluidic “double-cross” junction. Gel bead-containing droplets flow to a reservoir where gel beads are dissolved, initiating whole genome primer extension. The products are pooled from each droplet. The final library preparation requires shearing the libraries and incorporation of Illumina adapters. (b) Top panel, linked-reads of the ALK gene from the NA12878 WGS sample. Each line represents linked-reads with the same barcode, with dots representing reads, and color depicting reads with different barcodes. Middle panel, blue blocks showing exon boundaries of the ALK gene. Bottom panel, linked-reads of ALK gene from the NA12878 exome data. Although there are only reads in exon regions, reads from neighboring exons are linked because of common barcodes. Only a very small fraction of linked-reads is presented here to conserve space.
Figure Legend Snippet: Overview of the technology for generating linked-reads (a) Gel beads loaded with primers and barcoded oligonucleotides are first mixed with DNA and enzyme mixture, and subsequently mixed with oil-surfactant solution at a microfluidic “double-cross” junction. Gel bead-containing droplets flow to a reservoir where gel beads are dissolved, initiating whole genome primer extension. The products are pooled from each droplet. The final library preparation requires shearing the libraries and incorporation of Illumina adapters. (b) Top panel, linked-reads of the ALK gene from the NA12878 WGS sample. Each line represents linked-reads with the same barcode, with dots representing reads, and color depicting reads with different barcodes. Middle panel, blue blocks showing exon boundaries of the ALK gene. Bottom panel, linked-reads of ALK gene from the NA12878 exome data. Although there are only reads in exon regions, reads from neighboring exons are linked because of common barcodes. Only a very small fraction of linked-reads is presented here to conserve space.

Techniques Used: Flow Cytometry

27) Product Images from "Pro-oncogenic Roles of HLXB9 Protein in Insulinoma Cells through Interaction with Nono Protein and Down-regulation of the c-Met Inhibitor Cblb (Casitas B-lineage Lymphoma b) *"

Article Title: Pro-oncogenic Roles of HLXB9 Protein in Insulinoma Cells through Interaction with Nono Protein and Down-regulation of the c-Met Inhibitor Cblb (Casitas B-lineage Lymphoma b) *

Journal: The Journal of Biological Chemistry

doi: 10.1074/jbc.M115.661413

HLXB9 binding motif in the Cblb promoter. A , consensus motif in anti-HB9-PO4 ChIP-Seq tags located at promoter regions. De novo ). B , sequence of the HLXB9 binding motifs in the Cblb promoter. The top line shows the putative HLXB9 binding motifs ( red ) in the DNA sequence of the Cblb promoter (−741 to −710 region from the transcriptional start site is shown). The bottom line shows nucleotide substitutions ( blue ) to mutate the motifs by site-directed mutagenesis in the Cblb-promoter construct used in C. C and D , HLXB9 did not suppress the activity of the Cblb promoter containing mutations at the HLXB9 binding motifs. The putative HLXB9 binding motifs shown in B were mutated by site-directed mutagenesis of the PG02-Cblb promoter construct and analyzed for promoter activity in MIN6-4N cells. RLU for each of the transfections are shown. Compared with the empty vector PG02, the PG02-Cblb-SDM2 plasmid showed significantly high RLU and co-expression of increasing amounts of HLXB9 did not suppress the Cblb promoter activity. Error bar = Mean and S.D. from 3 experiments, * = p
Figure Legend Snippet: HLXB9 binding motif in the Cblb promoter. A , consensus motif in anti-HB9-PO4 ChIP-Seq tags located at promoter regions. De novo ). B , sequence of the HLXB9 binding motifs in the Cblb promoter. The top line shows the putative HLXB9 binding motifs ( red ) in the DNA sequence of the Cblb promoter (−741 to −710 region from the transcriptional start site is shown). The bottom line shows nucleotide substitutions ( blue ) to mutate the motifs by site-directed mutagenesis in the Cblb-promoter construct used in C. C and D , HLXB9 did not suppress the activity of the Cblb promoter containing mutations at the HLXB9 binding motifs. The putative HLXB9 binding motifs shown in B were mutated by site-directed mutagenesis of the PG02-Cblb promoter construct and analyzed for promoter activity in MIN6-4N cells. RLU for each of the transfections are shown. Compared with the empty vector PG02, the PG02-Cblb-SDM2 plasmid showed significantly high RLU and co-expression of increasing amounts of HLXB9 did not suppress the Cblb promoter activity. Error bar = Mean and S.D. from 3 experiments, * = p

Techniques Used: Binding Assay, Chromatin Immunoprecipitation, Sequencing, Mutagenesis, Construct, Activity Assay, Transfection, Plasmid Preparation, Expressing

HLXB9 co-localizes with Nono in the nucleus, and co-overexpression of HLXB9 and Nono decreases the overexpression of Nono with translocation of HLXB9 into the cytoplasm. A , endogenous phospho-HLXB9 co-localizes with endogenous Nono in the nucleus. IF images of MIN6-4N cells show endogenous Nono ( red ) and phospho-HLXB9 ( green ). DAPI was used to detect the nuclei ( blue ). A merged image of the red and green IF shows co-localization of Nono and phospho-HLXB9 in subnuclear spots and some regions with phospho-HLXB9 ( green ) that did not co-localize with Nono. B , overexpression of HLXB9 decreases the level of overexpressed Nono protein. Western blots are shown of WCE and the pellet leftover after WCE preparation from MIN6-4N cells expressing mh-HB9-WT, FLAG-Nono, or both together. Empty vector DNA ( Vec ) was used to maintain the same amount of DNA in the transfections. The expression of transfected HLXB9 was detected with the anti-myc-tag; Nono was detected with the anti-FLAG-tag and with anti-Nono to detect both endogenous and transfected FLAG-tagged Nono. β-Actin was used as the loading control. Endogenous Nono levels were not affected by HLXB9 overexpression. However, the level of transfected FLAG-Nono was reduced upon HLXB9 overexpression. A similar pattern of bands was seen in the pellet leftover after WCE preparation, indicating that the reduced level of Nono upon HLXB9 overexpression was not due to differential cell lysis in the WCE preparation. C , HLXB9 did not reduce the expression of endogenous Nono protein. Western blot analysis to detect endogenous HLXB9 and Nono using WCE prepared from MIN6-4N cells transfected with control siRNA ( siC ) or HLXB9 siRNA ( siHB9 ) is shown. p84 was used as the loading control. HLXB9 was significantly knocked down, but that did not affect the level of endogenous Nono. D , co-overexpression of HLXB9 and Nono reduces the level of Nono protein in the nucleus with translocation of HLXB9 to the cytoplasm. Shown is Western blot analysis of subcellular fractionation of CE, NE, and CB/PE from MIN6-4N cells expressing mh-HB9-WT, FLAG-Nono, or both together. Empty vector DNA ( Vec ) was used to maintain the same amount of DNA in the transfections. The expression of transfected HLXB9 was detected with the anti-myc-tag; Nono was detected with the anti-FLAG-tag; detection of marker proteins (Hsp90 for CE, p84 for NE, and histone H3 for CB/PE) showed minimal cross-contamination of the fractions and also served as loading controls for each fraction. Overexpressed Nono was found in the NE and in the CB/PE, but its level in NE was reduced by HLXB9 overexpression. Overexpressed HLXB9 was mostly located in the CB/PE and with a significant amount in the nucleus, but it was also detected in the CE by Nono overexpression.
Figure Legend Snippet: HLXB9 co-localizes with Nono in the nucleus, and co-overexpression of HLXB9 and Nono decreases the overexpression of Nono with translocation of HLXB9 into the cytoplasm. A , endogenous phospho-HLXB9 co-localizes with endogenous Nono in the nucleus. IF images of MIN6-4N cells show endogenous Nono ( red ) and phospho-HLXB9 ( green ). DAPI was used to detect the nuclei ( blue ). A merged image of the red and green IF shows co-localization of Nono and phospho-HLXB9 in subnuclear spots and some regions with phospho-HLXB9 ( green ) that did not co-localize with Nono. B , overexpression of HLXB9 decreases the level of overexpressed Nono protein. Western blots are shown of WCE and the pellet leftover after WCE preparation from MIN6-4N cells expressing mh-HB9-WT, FLAG-Nono, or both together. Empty vector DNA ( Vec ) was used to maintain the same amount of DNA in the transfections. The expression of transfected HLXB9 was detected with the anti-myc-tag; Nono was detected with the anti-FLAG-tag and with anti-Nono to detect both endogenous and transfected FLAG-tagged Nono. β-Actin was used as the loading control. Endogenous Nono levels were not affected by HLXB9 overexpression. However, the level of transfected FLAG-Nono was reduced upon HLXB9 overexpression. A similar pattern of bands was seen in the pellet leftover after WCE preparation, indicating that the reduced level of Nono upon HLXB9 overexpression was not due to differential cell lysis in the WCE preparation. C , HLXB9 did not reduce the expression of endogenous Nono protein. Western blot analysis to detect endogenous HLXB9 and Nono using WCE prepared from MIN6-4N cells transfected with control siRNA ( siC ) or HLXB9 siRNA ( siHB9 ) is shown. p84 was used as the loading control. HLXB9 was significantly knocked down, but that did not affect the level of endogenous Nono. D , co-overexpression of HLXB9 and Nono reduces the level of Nono protein in the nucleus with translocation of HLXB9 to the cytoplasm. Shown is Western blot analysis of subcellular fractionation of CE, NE, and CB/PE from MIN6-4N cells expressing mh-HB9-WT, FLAG-Nono, or both together. Empty vector DNA ( Vec ) was used to maintain the same amount of DNA in the transfections. The expression of transfected HLXB9 was detected with the anti-myc-tag; Nono was detected with the anti-FLAG-tag; detection of marker proteins (Hsp90 for CE, p84 for NE, and histone H3 for CB/PE) showed minimal cross-contamination of the fractions and also served as loading controls for each fraction. Overexpressed Nono was found in the NE and in the CB/PE, but its level in NE was reduced by HLXB9 overexpression. Overexpressed HLXB9 was mostly located in the CB/PE and with a significant amount in the nucleus, but it was also detected in the CE by Nono overexpression.

Techniques Used: Over Expression, Translocation Assay, Western Blot, Expressing, Plasmid Preparation, Transfection, FLAG-tag, Lysis, Fractionation, Marker

Identification of Cblb as a phospho-HLXB9 target gene. A , the anti-HB9-PO4 antibody specifically recognizes the phosphorylated isoform of HLXB9. WCE and chromatin were prepared from MIN6-4N cells transfected with a plasmid expressing myc-his-tagged HLXB9 ( mh-HB9-WT ). WCE was used IP with rabbit antibodies anti-myc-tag or anti-HB9-PO4 and detected by Western blot ( WB ) with mouse anti-myc-tag. Rabbit anti-HA-tag was used as the negative control. The input WCE and anti-myc-tag IP display a doublet corresponding to phospho-HLXB9 and unphosphorylated HLXB9. Anti-HB9-PO4 could specifically immunoprecipitate phospho-HLXB9 corresponding to the top band of the doublet. B , significant enrichment of promoter regions among the anti-HB9-PO4 ChIP-Seq tags. Chromatin prepared from MIN6-4N cells transfected in A was used for ChIP with anti-HB9-PO4. DNA obtained before and after ChIP was used for preparing libraries followed by deep sequencing (ChIP-Seq) and mapping of the anti-HB9-PO4-specific ChIP-Seq tags to the mouse genome. The pie chart shows the percent distribution of tags in the mouse genome (a typical input library, Genomatix) and in the anti-HB9-PO4 ChIP-Seq at the indicated regions; 20% of the anti-HB9-PO4 ChIP-Seq tags were located near promoter regions and selected for further analysis. C , phospho-HLXB9 occupancy is highest at the Arid1b and Cblb gene in cells overexpressing HLXB9. ChIP-quantitative PCR assay for validating the 10 phospho-HLXB9 targets is shown as the percent of input chromatin DNA PCR for each primer pair. Chromatin prepared from MIN6-4N cells expressing mh-HB9-WT was used for anti-HB9-PO4 ChIP. Also shown is a Western blot confirming overexpression of HLXB9 (myc-tag antibody) and β-actin as the loading control. D , endogenous phospho-HLXB9 occupancy is highest at the Cblb gene. ChIP-quantitative PCR assay of the 10 phospho-HLXB9 targets is shown as percent of input chromatin DNA PCR for each primer pair. Chromatin prepared from MIN6-4N cells was used for endogenous anti-HB9-PO4 ChIP. Endogenous phospho-HLXB9 occupancy was only detected at Cblb. E and F , H3K4me3 at Cblb unaffected but reduced H3K27me3 upon HLXB9 knockdown. ChIP-quantitative PCR assay of the 10 phospho-HLXB9 targets is shown as the percent of input chromatin DNA PCR for each primer pair. Chromatin prepared from MIN6-4N cells transfected with control siRNA ( siC ) or HLXB9 siRNA ( siHB9 ) was used for endogenous anti-H3K4me3 ChIP ( E ) or H3K27me3 ChIP ( F ). Also shown is a Western blot confirming knockdown of HLXB9 (HLXB9 antibody) and β-actin as the loading control. In siC versus siHB9, reciprocal H3K4me3 or H3K27me3 at only Cblb was HLXB9 binding-dependent because endogenous phospho-HLXB9 was only found to occupy Cblb ( D ).
Figure Legend Snippet: Identification of Cblb as a phospho-HLXB9 target gene. A , the anti-HB9-PO4 antibody specifically recognizes the phosphorylated isoform of HLXB9. WCE and chromatin were prepared from MIN6-4N cells transfected with a plasmid expressing myc-his-tagged HLXB9 ( mh-HB9-WT ). WCE was used IP with rabbit antibodies anti-myc-tag or anti-HB9-PO4 and detected by Western blot ( WB ) with mouse anti-myc-tag. Rabbit anti-HA-tag was used as the negative control. The input WCE and anti-myc-tag IP display a doublet corresponding to phospho-HLXB9 and unphosphorylated HLXB9. Anti-HB9-PO4 could specifically immunoprecipitate phospho-HLXB9 corresponding to the top band of the doublet. B , significant enrichment of promoter regions among the anti-HB9-PO4 ChIP-Seq tags. Chromatin prepared from MIN6-4N cells transfected in A was used for ChIP with anti-HB9-PO4. DNA obtained before and after ChIP was used for preparing libraries followed by deep sequencing (ChIP-Seq) and mapping of the anti-HB9-PO4-specific ChIP-Seq tags to the mouse genome. The pie chart shows the percent distribution of tags in the mouse genome (a typical input library, Genomatix) and in the anti-HB9-PO4 ChIP-Seq at the indicated regions; 20% of the anti-HB9-PO4 ChIP-Seq tags were located near promoter regions and selected for further analysis. C , phospho-HLXB9 occupancy is highest at the Arid1b and Cblb gene in cells overexpressing HLXB9. ChIP-quantitative PCR assay for validating the 10 phospho-HLXB9 targets is shown as the percent of input chromatin DNA PCR for each primer pair. Chromatin prepared from MIN6-4N cells expressing mh-HB9-WT was used for anti-HB9-PO4 ChIP. Also shown is a Western blot confirming overexpression of HLXB9 (myc-tag antibody) and β-actin as the loading control. D , endogenous phospho-HLXB9 occupancy is highest at the Cblb gene. ChIP-quantitative PCR assay of the 10 phospho-HLXB9 targets is shown as percent of input chromatin DNA PCR for each primer pair. Chromatin prepared from MIN6-4N cells was used for endogenous anti-HB9-PO4 ChIP. Endogenous phospho-HLXB9 occupancy was only detected at Cblb. E and F , H3K4me3 at Cblb unaffected but reduced H3K27me3 upon HLXB9 knockdown. ChIP-quantitative PCR assay of the 10 phospho-HLXB9 targets is shown as the percent of input chromatin DNA PCR for each primer pair. Chromatin prepared from MIN6-4N cells transfected with control siRNA ( siC ) or HLXB9 siRNA ( siHB9 ) was used for endogenous anti-H3K4me3 ChIP ( E ) or H3K27me3 ChIP ( F ). Also shown is a Western blot confirming knockdown of HLXB9 (HLXB9 antibody) and β-actin as the loading control. In siC versus siHB9, reciprocal H3K4me3 or H3K27me3 at only Cblb was HLXB9 binding-dependent because endogenous phospho-HLXB9 was only found to occupy Cblb ( D ).

Techniques Used: Transfection, Plasmid Preparation, Expressing, Western Blot, Negative Control, Chromatin Immunoprecipitation, Sequencing, Real-time Polymerase Chain Reaction, Polymerase Chain Reaction, Over Expression, Binding Assay

28) Product Images from "Shallow whole genome sequencing for robust copy number profiling of formalin-fixed paraffin-embedded breast cancers"

Article Title: Shallow whole genome sequencing for robust copy number profiling of formalin-fixed paraffin-embedded breast cancers

Journal: Experimental and Molecular Pathology

doi: 10.1016/j.yexmp.2018.03.006

Features of input DNA and libraries generated from blocks less and more than five years. Dot plots represent the range (minimum-maximum) of observed values for each of the following categories and the red dot represents the median. A. The quality of input DNA inferred by ΔCt. B. Fragment sizes of the libraries in base pair. C. The library yield in nanomoles.
Figure Legend Snippet: Features of input DNA and libraries generated from blocks less and more than five years. Dot plots represent the range (minimum-maximum) of observed values for each of the following categories and the red dot represents the median. A. The quality of input DNA inferred by ΔCt. B. Fragment sizes of the libraries in base pair. C. The library yield in nanomoles.

Techniques Used: Generated

Features of the sequencing libraries. Boxplots showing different features of input DNA and library yield relative to the different library qualities. A. Input DNA. B. Quality of input DNA inferred from ΔCt. C. Fragment size of libraries. D. Library yield. VG = very good, G = good, I = intermediate, P = poor, F = fail.
Figure Legend Snippet: Features of the sequencing libraries. Boxplots showing different features of input DNA and library yield relative to the different library qualities. A. Input DNA. B. Quality of input DNA inferred from ΔCt. C. Fragment size of libraries. D. Library yield. VG = very good, G = good, I = intermediate, P = poor, F = fail.

Techniques Used: Sequencing

29) Product Images from "A high-throughput ChIP-Seq for large-scale chromatin studies"

Article Title: A high-throughput ChIP-Seq for large-scale chromatin studies

Journal: Molecular Systems Biology

doi: 10.15252/msb.20145776

Comparative representation of the ChIP-Seq and Bar-ChIP workflows Yeast cultures are crosslinked using formaldehyde. Chromatin is then extracted and fragmented using micrococcal nuclease (MNase) digestion. In a classical ChIP-Seq protocol, MNase-treated chromatin is directly immuno-precipitated with an antibody against the protein modification or factor of interest. Recovered DNA is then barcoded and used to generate an amplified DNA library ready for paired-end sequencing. In the Bar-ChIP protocol, fragmented chromatin is barcoded through ligation of molecular barcodes prior to immuno-precipitation. DNA recovered from the IP can directly be amplified by PCR using Illumina primers and deep-sequenced. The presence of the barcoding step early in the workflow allows for multiplexing of IP assays.
Figure Legend Snippet: Comparative representation of the ChIP-Seq and Bar-ChIP workflows Yeast cultures are crosslinked using formaldehyde. Chromatin is then extracted and fragmented using micrococcal nuclease (MNase) digestion. In a classical ChIP-Seq protocol, MNase-treated chromatin is directly immuno-precipitated with an antibody against the protein modification or factor of interest. Recovered DNA is then barcoded and used to generate an amplified DNA library ready for paired-end sequencing. In the Bar-ChIP protocol, fragmented chromatin is barcoded through ligation of molecular barcodes prior to immuno-precipitation. DNA recovered from the IP can directly be amplified by PCR using Illumina primers and deep-sequenced. The presence of the barcoding step early in the workflow allows for multiplexing of IP assays.

Techniques Used: Chromatin Immunoprecipitation, Modification, Amplification, Sequencing, Ligation, Immunoprecipitation, Polymerase Chain Reaction, Multiplexing

Highly multiplexed ChIP experiment based on the Bar-ChIP protocol Schematic representation of the experimental design. Cultures corresponding to distinct yeast strains were harvested, crosslinked and their MNase-treated chromatin was barcoded to enable sample tracking. Aliquots from each of the barcoded chromatin samples were pooled together prior to immuno-precipitation against the histone modifications of interest. DNA recovered from each IP was amplified and sequenced using paired-end technology. Finally, barcode sequences were used to demultiplex sequencing datasets and attribute each read to the proper biological sample. Normalized proportion of reads attributed to each strain. One sequencing lane corresponded to multiplexed samples submitted to one IP assay. For each sequencing lane, read counts attributed to each biological sample were first divided by the total number of reads recovered from the lane ( Supplementary Fig. S6 ). For each biological sample, the resulting ratio was normalized using the proportion of reads in the chromatin input lane that was attributed to that biological sample to correct for biases in the initial pooling of fragmented chromatin samples. Note that each set1Δ library represents less than 0.17% of the total number of reads recovered for the IPs against H3K4 methylation while the set2Δ libraries represent not more than 0.8% of the total reads recovered for the IP against H3K36me3. Absolute numbers for recovered sequencing reads are indicated in Supplementary Fig. S7 .
Figure Legend Snippet: Highly multiplexed ChIP experiment based on the Bar-ChIP protocol Schematic representation of the experimental design. Cultures corresponding to distinct yeast strains were harvested, crosslinked and their MNase-treated chromatin was barcoded to enable sample tracking. Aliquots from each of the barcoded chromatin samples were pooled together prior to immuno-precipitation against the histone modifications of interest. DNA recovered from each IP was amplified and sequenced using paired-end technology. Finally, barcode sequences were used to demultiplex sequencing datasets and attribute each read to the proper biological sample. Normalized proportion of reads attributed to each strain. One sequencing lane corresponded to multiplexed samples submitted to one IP assay. For each sequencing lane, read counts attributed to each biological sample were first divided by the total number of reads recovered from the lane ( Supplementary Fig. S6 ). For each biological sample, the resulting ratio was normalized using the proportion of reads in the chromatin input lane that was attributed to that biological sample to correct for biases in the initial pooling of fragmented chromatin samples. Note that each set1Δ library represents less than 0.17% of the total number of reads recovered for the IPs against H3K4 methylation while the set2Δ libraries represent not more than 0.8% of the total reads recovered for the IP against H3K36me3. Absolute numbers for recovered sequencing reads are indicated in Supplementary Fig. S7 .

Techniques Used: Chromatin Immunoprecipitation, Immunoprecipitation, Amplification, Sequencing, Methylation

30) Product Images from "Pro-oncogenic Roles of HLXB9 Protein in Insulinoma Cells through Interaction with Nono Protein and Down-regulation of the c-Met Inhibitor Cblb (Casitas B-lineage Lymphoma b) *"

Article Title: Pro-oncogenic Roles of HLXB9 Protein in Insulinoma Cells through Interaction with Nono Protein and Down-regulation of the c-Met Inhibitor Cblb (Casitas B-lineage Lymphoma b) *

Journal: The Journal of Biological Chemistry

doi: 10.1074/jbc.M115.661413

HLXB9 binding motif in the Cblb promoter. A , consensus motif in anti-HB9-PO4 ChIP-Seq tags located at promoter regions. De novo ). B , sequence of the HLXB9 binding motifs in the Cblb promoter. The top line shows the putative HLXB9 binding motifs ( red ) in the DNA sequence of the Cblb promoter (−741 to −710 region from the transcriptional start site is shown). The bottom line shows nucleotide substitutions ( blue ) to mutate the motifs by site-directed mutagenesis in the Cblb-promoter construct used in C. C and D , HLXB9 did not suppress the activity of the Cblb promoter containing mutations at the HLXB9 binding motifs. The putative HLXB9 binding motifs shown in B were mutated by site-directed mutagenesis of the PG02-Cblb promoter construct and analyzed for promoter activity in MIN6-4N cells. RLU for each of the transfections are shown. Compared with the empty vector PG02, the PG02-Cblb-SDM2 plasmid showed significantly high RLU and co-expression of increasing amounts of HLXB9 did not suppress the Cblb promoter activity. Error bar = Mean and S.D. from 3 experiments, * = p
Figure Legend Snippet: HLXB9 binding motif in the Cblb promoter. A , consensus motif in anti-HB9-PO4 ChIP-Seq tags located at promoter regions. De novo ). B , sequence of the HLXB9 binding motifs in the Cblb promoter. The top line shows the putative HLXB9 binding motifs ( red ) in the DNA sequence of the Cblb promoter (−741 to −710 region from the transcriptional start site is shown). The bottom line shows nucleotide substitutions ( blue ) to mutate the motifs by site-directed mutagenesis in the Cblb-promoter construct used in C. C and D , HLXB9 did not suppress the activity of the Cblb promoter containing mutations at the HLXB9 binding motifs. The putative HLXB9 binding motifs shown in B were mutated by site-directed mutagenesis of the PG02-Cblb promoter construct and analyzed for promoter activity in MIN6-4N cells. RLU for each of the transfections are shown. Compared with the empty vector PG02, the PG02-Cblb-SDM2 plasmid showed significantly high RLU and co-expression of increasing amounts of HLXB9 did not suppress the Cblb promoter activity. Error bar = Mean and S.D. from 3 experiments, * = p

Techniques Used: Binding Assay, Chromatin Immunoprecipitation, Sequencing, Mutagenesis, Construct, Activity Assay, Transfection, Plasmid Preparation, Expressing

HLXB9 co-localizes with Nono in the nucleus, and co-overexpression of HLXB9 and Nono decreases the overexpression of Nono with translocation of HLXB9 into the cytoplasm. A , endogenous phospho-HLXB9 co-localizes with endogenous Nono in the nucleus. IF images of MIN6-4N cells show endogenous Nono ( red ) and phospho-HLXB9 ( green ). DAPI was used to detect the nuclei ( blue ). A merged image of the red and green IF shows co-localization of Nono and phospho-HLXB9 in subnuclear spots and some regions with phospho-HLXB9 ( green ) that did not co-localize with Nono. B , overexpression of HLXB9 decreases the level of overexpressed Nono protein. Western blots are shown of WCE and the pellet leftover after WCE preparation from MIN6-4N cells expressing mh-HB9-WT, FLAG-Nono, or both together. Empty vector DNA ( Vec ) was used to maintain the same amount of DNA in the transfections. The expression of transfected HLXB9 was detected with the anti-myc-tag; Nono was detected with the anti-FLAG-tag and with anti-Nono to detect both endogenous and transfected FLAG-tagged Nono. β-Actin was used as the loading control. Endogenous Nono levels were not affected by HLXB9 overexpression. However, the level of transfected FLAG-Nono was reduced upon HLXB9 overexpression. A similar pattern of bands was seen in the pellet leftover after WCE preparation, indicating that the reduced level of Nono upon HLXB9 overexpression was not due to differential cell lysis in the WCE preparation. C , HLXB9 did not reduce the expression of endogenous Nono protein. Western blot analysis to detect endogenous HLXB9 and Nono using WCE prepared from MIN6-4N cells transfected with control siRNA ( siC ) or HLXB9 siRNA ( siHB9 ) is shown. p84 was used as the loading control. HLXB9 was significantly knocked down, but that did not affect the level of endogenous Nono. D , co-overexpression of HLXB9 and Nono reduces the level of Nono protein in the nucleus with translocation of HLXB9 to the cytoplasm. Shown is Western blot analysis of subcellular fractionation of CE, NE, and CB/PE from MIN6-4N cells expressing mh-HB9-WT, FLAG-Nono, or both together. Empty vector DNA ( Vec ) was used to maintain the same amount of DNA in the transfections. The expression of transfected HLXB9 was detected with the anti-myc-tag; Nono was detected with the anti-FLAG-tag; detection of marker proteins (Hsp90 for CE, p84 for NE, and histone H3 for CB/PE) showed minimal cross-contamination of the fractions and also served as loading controls for each fraction. Overexpressed Nono was found in the NE and in the CB/PE, but its level in NE was reduced by HLXB9 overexpression. Overexpressed HLXB9 was mostly located in the CB/PE and with a significant amount in the nucleus, but it was also detected in the CE by Nono overexpression.
Figure Legend Snippet: HLXB9 co-localizes with Nono in the nucleus, and co-overexpression of HLXB9 and Nono decreases the overexpression of Nono with translocation of HLXB9 into the cytoplasm. A , endogenous phospho-HLXB9 co-localizes with endogenous Nono in the nucleus. IF images of MIN6-4N cells show endogenous Nono ( red ) and phospho-HLXB9 ( green ). DAPI was used to detect the nuclei ( blue ). A merged image of the red and green IF shows co-localization of Nono and phospho-HLXB9 in subnuclear spots and some regions with phospho-HLXB9 ( green ) that did not co-localize with Nono. B , overexpression of HLXB9 decreases the level of overexpressed Nono protein. Western blots are shown of WCE and the pellet leftover after WCE preparation from MIN6-4N cells expressing mh-HB9-WT, FLAG-Nono, or both together. Empty vector DNA ( Vec ) was used to maintain the same amount of DNA in the transfections. The expression of transfected HLXB9 was detected with the anti-myc-tag; Nono was detected with the anti-FLAG-tag and with anti-Nono to detect both endogenous and transfected FLAG-tagged Nono. β-Actin was used as the loading control. Endogenous Nono levels were not affected by HLXB9 overexpression. However, the level of transfected FLAG-Nono was reduced upon HLXB9 overexpression. A similar pattern of bands was seen in the pellet leftover after WCE preparation, indicating that the reduced level of Nono upon HLXB9 overexpression was not due to differential cell lysis in the WCE preparation. C , HLXB9 did not reduce the expression of endogenous Nono protein. Western blot analysis to detect endogenous HLXB9 and Nono using WCE prepared from MIN6-4N cells transfected with control siRNA ( siC ) or HLXB9 siRNA ( siHB9 ) is shown. p84 was used as the loading control. HLXB9 was significantly knocked down, but that did not affect the level of endogenous Nono. D , co-overexpression of HLXB9 and Nono reduces the level of Nono protein in the nucleus with translocation of HLXB9 to the cytoplasm. Shown is Western blot analysis of subcellular fractionation of CE, NE, and CB/PE from MIN6-4N cells expressing mh-HB9-WT, FLAG-Nono, or both together. Empty vector DNA ( Vec ) was used to maintain the same amount of DNA in the transfections. The expression of transfected HLXB9 was detected with the anti-myc-tag; Nono was detected with the anti-FLAG-tag; detection of marker proteins (Hsp90 for CE, p84 for NE, and histone H3 for CB/PE) showed minimal cross-contamination of the fractions and also served as loading controls for each fraction. Overexpressed Nono was found in the NE and in the CB/PE, but its level in NE was reduced by HLXB9 overexpression. Overexpressed HLXB9 was mostly located in the CB/PE and with a significant amount in the nucleus, but it was also detected in the CE by Nono overexpression.

Techniques Used: Over Expression, Translocation Assay, Western Blot, Expressing, Plasmid Preparation, Transfection, FLAG-tag, Lysis, Fractionation, Marker

Identification of Cblb as a phospho-HLXB9 target gene. A , the anti-HB9-PO4 antibody specifically recognizes the phosphorylated isoform of HLXB9. WCE and chromatin were prepared from MIN6-4N cells transfected with a plasmid expressing myc-his-tagged HLXB9 ( mh-HB9-WT ). WCE was used IP with rabbit antibodies anti-myc-tag or anti-HB9-PO4 and detected by Western blot ( WB ) with mouse anti-myc-tag. Rabbit anti-HA-tag was used as the negative control. The input WCE and anti-myc-tag IP display a doublet corresponding to phospho-HLXB9 and unphosphorylated HLXB9. Anti-HB9-PO4 could specifically immunoprecipitate phospho-HLXB9 corresponding to the top band of the doublet. B , significant enrichment of promoter regions among the anti-HB9-PO4 ChIP-Seq tags. Chromatin prepared from MIN6-4N cells transfected in A was used for ChIP with anti-HB9-PO4. DNA obtained before and after ChIP was used for preparing libraries followed by deep sequencing (ChIP-Seq) and mapping of the anti-HB9-PO4-specific ChIP-Seq tags to the mouse genome. The pie chart shows the percent distribution of tags in the mouse genome (a typical input library, Genomatix) and in the anti-HB9-PO4 ChIP-Seq at the indicated regions; 20% of the anti-HB9-PO4 ChIP-Seq tags were located near promoter regions and selected for further analysis. C , phospho-HLXB9 occupancy is highest at the Arid1b and Cblb gene in cells overexpressing HLXB9. ChIP-quantitative PCR assay for validating the 10 phospho-HLXB9 targets is shown as the percent of input chromatin DNA PCR for each primer pair. Chromatin prepared from MIN6-4N cells expressing mh-HB9-WT was used for anti-HB9-PO4 ChIP. Also shown is a Western blot confirming overexpression of HLXB9 (myc-tag antibody) and β-actin as the loading control. D , endogenous phospho-HLXB9 occupancy is highest at the Cblb gene. ChIP-quantitative PCR assay of the 10 phospho-HLXB9 targets is shown as percent of input chromatin DNA PCR for each primer pair. Chromatin prepared from MIN6-4N cells was used for endogenous anti-HB9-PO4 ChIP. Endogenous phospho-HLXB9 occupancy was only detected at Cblb. E and F , H3K4me3 at Cblb unaffected but reduced H3K27me3 upon HLXB9 knockdown. ChIP-quantitative PCR assay of the 10 phospho-HLXB9 targets is shown as the percent of input chromatin DNA PCR for each primer pair. Chromatin prepared from MIN6-4N cells transfected with control siRNA ( siC ) or HLXB9 siRNA ( siHB9 ) was used for endogenous anti-H3K4me3 ChIP ( E ) or H3K27me3 ChIP ( F ). Also shown is a Western blot confirming knockdown of HLXB9 (HLXB9 antibody) and β-actin as the loading control. In siC versus siHB9, reciprocal H3K4me3 or H3K27me3 at only Cblb was HLXB9 binding-dependent because endogenous phospho-HLXB9 was only found to occupy Cblb ( D ).
Figure Legend Snippet: Identification of Cblb as a phospho-HLXB9 target gene. A , the anti-HB9-PO4 antibody specifically recognizes the phosphorylated isoform of HLXB9. WCE and chromatin were prepared from MIN6-4N cells transfected with a plasmid expressing myc-his-tagged HLXB9 ( mh-HB9-WT ). WCE was used IP with rabbit antibodies anti-myc-tag or anti-HB9-PO4 and detected by Western blot ( WB ) with mouse anti-myc-tag. Rabbit anti-HA-tag was used as the negative control. The input WCE and anti-myc-tag IP display a doublet corresponding to phospho-HLXB9 and unphosphorylated HLXB9. Anti-HB9-PO4 could specifically immunoprecipitate phospho-HLXB9 corresponding to the top band of the doublet. B , significant enrichment of promoter regions among the anti-HB9-PO4 ChIP-Seq tags. Chromatin prepared from MIN6-4N cells transfected in A was used for ChIP with anti-HB9-PO4. DNA obtained before and after ChIP was used for preparing libraries followed by deep sequencing (ChIP-Seq) and mapping of the anti-HB9-PO4-specific ChIP-Seq tags to the mouse genome. The pie chart shows the percent distribution of tags in the mouse genome (a typical input library, Genomatix) and in the anti-HB9-PO4 ChIP-Seq at the indicated regions; 20% of the anti-HB9-PO4 ChIP-Seq tags were located near promoter regions and selected for further analysis. C , phospho-HLXB9 occupancy is highest at the Arid1b and Cblb gene in cells overexpressing HLXB9. ChIP-quantitative PCR assay for validating the 10 phospho-HLXB9 targets is shown as the percent of input chromatin DNA PCR for each primer pair. Chromatin prepared from MIN6-4N cells expressing mh-HB9-WT was used for anti-HB9-PO4 ChIP. Also shown is a Western blot confirming overexpression of HLXB9 (myc-tag antibody) and β-actin as the loading control. D , endogenous phospho-HLXB9 occupancy is highest at the Cblb gene. ChIP-quantitative PCR assay of the 10 phospho-HLXB9 targets is shown as percent of input chromatin DNA PCR for each primer pair. Chromatin prepared from MIN6-4N cells was used for endogenous anti-HB9-PO4 ChIP. Endogenous phospho-HLXB9 occupancy was only detected at Cblb. E and F , H3K4me3 at Cblb unaffected but reduced H3K27me3 upon HLXB9 knockdown. ChIP-quantitative PCR assay of the 10 phospho-HLXB9 targets is shown as the percent of input chromatin DNA PCR for each primer pair. Chromatin prepared from MIN6-4N cells transfected with control siRNA ( siC ) or HLXB9 siRNA ( siHB9 ) was used for endogenous anti-H3K4me3 ChIP ( E ) or H3K27me3 ChIP ( F ). Also shown is a Western blot confirming knockdown of HLXB9 (HLXB9 antibody) and β-actin as the loading control. In siC versus siHB9, reciprocal H3K4me3 or H3K27me3 at only Cblb was HLXB9 binding-dependent because endogenous phospho-HLXB9 was only found to occupy Cblb ( D ).

Techniques Used: Transfection, Plasmid Preparation, Expressing, Western Blot, Negative Control, Chromatin Immunoprecipitation, Sequencing, Real-time Polymerase Chain Reaction, Polymerase Chain Reaction, Over Expression, Binding Assay

31) Product Images from "RNAi-independent role for Argonaute2 in CTCF/CP190 chromatin insulator function"

Article Title: RNAi-independent role for Argonaute2 in CTCF/CP190 chromatin insulator function

Journal: Genes & Development

doi: 10.1101/gad.16651211

AGO2 is required for looping interactions throughout the Abd-B locus and proper gene expression. ( A ) CTCF and CP190 chromatin association is unaffected in AGO2 51B -null mutants. Adult heads of wild type (blue) as well as AGO2 51B /+ (red) or AGO2 51B (green) derived from AGO2 51B germline clones were subjected to ChIP using α-CP190 and α-CTCF. Locations of primer sets are indicated in B . Percent input DNA immunoprecipitated is shown for each primer set, and error bars indicate standard deviation of quadruplicate PCR measurements. ( B ) 3C looping interactions between cis -regulatory elements of the Abd-B locus are dependent on AGO2 . Relative interaction frequencies between EcoRI restriction fragments (triangles) and anchor regions (red vertical lines) are shown for wild type (open circles) and CP190 P11 /CP190 H31-2 (filled red circles), CTCF y+2 (filled green circles), and AGO2 51B (filled orange circles) mutant larval brains and imaginal discs. ( C ) Western blotting of lysates from S3 cells mock-treated (lane 1 ) or transfected with AGO2 siRNA (lane 2 ). ( D ) AGO2 and CTCF are required for proper Abd-B expression. RT–PCR to detect a common region or isoforms of Abd-B transcripts relative to RpL32 in oligo(dT) primed cDNA of S3 cells mock-treated (blue), CTCF-depleted (green), and AGO2-depleted (orange). Quantitative SYBR green PCR was performed in quadruplicate using S3 genomic DNA as a standard to normalize for primer efficiencies. ( E ) Model for AGO2 function with respect to CTCF/CP190 chromatin insulator activity. Looping at the Abd-B locus between the Fab-8 insulator and Abd-B promoter is dependent on CTCF/CP190 insulator interactions. This specialized configuration promotes interactions between Fab-8 -associated cis -regulatory elements and the promoters to facilitate proper gene expression. AGO2 is recruited depending on CTCF/CP190 chromatin association and acts to either promote or stabilize looping interactions. Transfer of AGO2 to noninsulator sites may be achieved through CTCF/CP190-dependent looping interactions.
Figure Legend Snippet: AGO2 is required for looping interactions throughout the Abd-B locus and proper gene expression. ( A ) CTCF and CP190 chromatin association is unaffected in AGO2 51B -null mutants. Adult heads of wild type (blue) as well as AGO2 51B /+ (red) or AGO2 51B (green) derived from AGO2 51B germline clones were subjected to ChIP using α-CP190 and α-CTCF. Locations of primer sets are indicated in B . Percent input DNA immunoprecipitated is shown for each primer set, and error bars indicate standard deviation of quadruplicate PCR measurements. ( B ) 3C looping interactions between cis -regulatory elements of the Abd-B locus are dependent on AGO2 . Relative interaction frequencies between EcoRI restriction fragments (triangles) and anchor regions (red vertical lines) are shown for wild type (open circles) and CP190 P11 /CP190 H31-2 (filled red circles), CTCF y+2 (filled green circles), and AGO2 51B (filled orange circles) mutant larval brains and imaginal discs. ( C ) Western blotting of lysates from S3 cells mock-treated (lane 1 ) or transfected with AGO2 siRNA (lane 2 ). ( D ) AGO2 and CTCF are required for proper Abd-B expression. RT–PCR to detect a common region or isoforms of Abd-B transcripts relative to RpL32 in oligo(dT) primed cDNA of S3 cells mock-treated (blue), CTCF-depleted (green), and AGO2-depleted (orange). Quantitative SYBR green PCR was performed in quadruplicate using S3 genomic DNA as a standard to normalize for primer efficiencies. ( E ) Model for AGO2 function with respect to CTCF/CP190 chromatin insulator activity. Looping at the Abd-B locus between the Fab-8 insulator and Abd-B promoter is dependent on CTCF/CP190 insulator interactions. This specialized configuration promotes interactions between Fab-8 -associated cis -regulatory elements and the promoters to facilitate proper gene expression. AGO2 is recruited depending on CTCF/CP190 chromatin association and acts to either promote or stabilize looping interactions. Transfer of AGO2 to noninsulator sites may be achieved through CTCF/CP190-dependent looping interactions.

Techniques Used: Expressing, Derivative Assay, Clone Assay, Chromatin Immunoprecipitation, Immunoprecipitation, Standard Deviation, Polymerase Chain Reaction, Mutagenesis, Western Blot, Transfection, Reverse Transcription Polymerase Chain Reaction, SYBR Green Assay, Activity Assay

CP190 and CTCF are required for AGO2 chromatin association and looping interactions throughout the Abd-B locus. ( A ) Western blotting of lysates from S2 cells mock-treated (lanes 1 , 3 ) or transfected with CP190 (lane 2 ) or CTCF (lane 4 ) dsRNA. ( B ) S2 cells mock-treated (blue) or transfected with CP190 (red) or CTCF (green) dsRNA were subjected to ChIP using α-CP190, α-CTCF, α-AGO2, α-Pho, and α-Pc antibodies. Locations of primer sets are indicated in D . Percent input DNA immunoprecipitated is shown for each primer set, and error bars indicate standard deviation of quadruplicate PCR measurements. IgG-negative control immunoprecipitations for all sites yielded
Figure Legend Snippet: CP190 and CTCF are required for AGO2 chromatin association and looping interactions throughout the Abd-B locus. ( A ) Western blotting of lysates from S2 cells mock-treated (lanes 1 , 3 ) or transfected with CP190 (lane 2 ) or CTCF (lane 4 ) dsRNA. ( B ) S2 cells mock-treated (blue) or transfected with CP190 (red) or CTCF (green) dsRNA were subjected to ChIP using α-CP190, α-CTCF, α-AGO2, α-Pho, and α-Pc antibodies. Locations of primer sets are indicated in D . Percent input DNA immunoprecipitated is shown for each primer set, and error bars indicate standard deviation of quadruplicate PCR measurements. IgG-negative control immunoprecipitations for all sites yielded

Techniques Used: Western Blot, Transfection, Chromatin Immunoprecipitation, Immunoprecipitation, Standard Deviation, Polymerase Chain Reaction, Negative Control

32) Product Images from "Homopolymer tail-mediated ligation PCR: a streamlined and highly efficient method for DNA cloning and library construction"

Article Title: Homopolymer tail-mediated ligation PCR: a streamlined and highly efficient method for DNA cloning and library construction

Journal: BioTechniques

doi: 10.2144/000113981

Sensitivity of HTML-PCR using Vibrio cholerae genomic DNA
Figure Legend Snippet: Sensitivity of HTML-PCR using Vibrio cholerae genomic DNA

Techniques Used: Polymerase Chain Reaction

33) Product Images from "Two Pif1 Family DNA Helicases Cooperate in Centromere Replication and Segregation in Saccharomyces cerevisiae"

Article Title: Two Pif1 Family DNA Helicases Cooperate in Centromere Replication and Segregation in Saccharomyces cerevisiae

Journal: Genetics

doi: 10.1534/genetics.118.301710

ScPif1 promotes replication through centromeres but only in rrm3 Δ cells. DNA from asynchronous WT and mutant cells was analyzed by 2D gel electrophoresis and Southern blot hybridization. (A) Schematic of 2D gel signal of Mfe I-digested DNA using the probe indicated in (B) for the CEN6 fragment. Arrows mark the pauses at CEN6 along the arc of Y-shaped replication intermediates. The solid arrow indicates the pause produced from replication forks originating from ARS605 . The dashed arrow indicates the pause arising from forks originating from ARS606 . 1N indicates nonreplicating linear Mfe I fragments. 2N indicates near fully replicated MfeI fragments. (B) Schematic of the Mfe I fragment that contains CEN6 in relation to the flanking replication origins. Cross-hatch box indicates the position of the radiolabeled probe used for Southern blot analysis. (C–H) Southern blot analysis of 2D gels on CEN6 from cells with the following genotypes: (C) WT, (D) rrm3 Δ, (E) pif1 Δ, (F) rrm3 Δ pif1 Δ, (G) tof1 Δ, and (H) rrm3 Δ tof1 Δ. (I) Same as (A) except that schematic is of CEN11 on a Sac I fragment. (J) Same as (B) except that it shows CEN11 in relation to the flanking replication origins. (K–N) Southern blot of 2D gels from the following strains: (K) WT, (L) rrm3 Δ, (M) pif1 Δ, and (N) rrm3 Δ pif1 Δ. For both CEN6 and CEN11, the signal at the pause was quantified as in Tran et al. (2017) and normalized to the pause signal in WT cells to obtain the relative fold c hange. The average fold difference of mutant over WT from two or more independent biological replicates is shown in the upper right corner of each Southern blot. (O) Freshly dissected WT, pif1-m2 , rrm3 Δ, and pif1-m2 rrm3 Δ spore clones derived from a multiply heterozygous but otherwise isogenic diploid were streaked on YEPD plate at 30° for 2 days. (P) Representative FACS profiles for one of five spore clones from WT, pif1-m2 , rrm3 Δ, and pif1-m2 rrm3 Δ cells. (Q) Quantified FACs data from five independent biological replicates of each strain that were grown asynchronously at 30°. The percent of cells from each strain that are in G1, S, and G2 phase are indicated along with statistical significance relative to WT cells; indicated by black asterisks. Statistical significance of pif1-m2 rrm3 Δ relative to rrm3 Δ is indicated by red asterisks. * P ≤ 0.05, ** P ≤ 0.01, *** P ≤ 0.001, **** P ≤ 0.0001. 2D, two-dimensional; WT, wild-type; YEPD, YEP and dextrose.
Figure Legend Snippet: ScPif1 promotes replication through centromeres but only in rrm3 Δ cells. DNA from asynchronous WT and mutant cells was analyzed by 2D gel electrophoresis and Southern blot hybridization. (A) Schematic of 2D gel signal of Mfe I-digested DNA using the probe indicated in (B) for the CEN6 fragment. Arrows mark the pauses at CEN6 along the arc of Y-shaped replication intermediates. The solid arrow indicates the pause produced from replication forks originating from ARS605 . The dashed arrow indicates the pause arising from forks originating from ARS606 . 1N indicates nonreplicating linear Mfe I fragments. 2N indicates near fully replicated MfeI fragments. (B) Schematic of the Mfe I fragment that contains CEN6 in relation to the flanking replication origins. Cross-hatch box indicates the position of the radiolabeled probe used for Southern blot analysis. (C–H) Southern blot analysis of 2D gels on CEN6 from cells with the following genotypes: (C) WT, (D) rrm3 Δ, (E) pif1 Δ, (F) rrm3 Δ pif1 Δ, (G) tof1 Δ, and (H) rrm3 Δ tof1 Δ. (I) Same as (A) except that schematic is of CEN11 on a Sac I fragment. (J) Same as (B) except that it shows CEN11 in relation to the flanking replication origins. (K–N) Southern blot of 2D gels from the following strains: (K) WT, (L) rrm3 Δ, (M) pif1 Δ, and (N) rrm3 Δ pif1 Δ. For both CEN6 and CEN11, the signal at the pause was quantified as in Tran et al. (2017) and normalized to the pause signal in WT cells to obtain the relative fold c hange. The average fold difference of mutant over WT from two or more independent biological replicates is shown in the upper right corner of each Southern blot. (O) Freshly dissected WT, pif1-m2 , rrm3 Δ, and pif1-m2 rrm3 Δ spore clones derived from a multiply heterozygous but otherwise isogenic diploid were streaked on YEPD plate at 30° for 2 days. (P) Representative FACS profiles for one of five spore clones from WT, pif1-m2 , rrm3 Δ, and pif1-m2 rrm3 Δ cells. (Q) Quantified FACs data from five independent biological replicates of each strain that were grown asynchronously at 30°. The percent of cells from each strain that are in G1, S, and G2 phase are indicated along with statistical significance relative to WT cells; indicated by black asterisks. Statistical significance of pif1-m2 rrm3 Δ relative to rrm3 Δ is indicated by red asterisks. * P ≤ 0.05, ** P ≤ 0.01, *** P ≤ 0.001, **** P ≤ 0.0001. 2D, two-dimensional; WT, wild-type; YEPD, YEP and dextrose.

Techniques Used: Mutagenesis, Two-Dimensional Gel Electrophoresis, Electrophoresis, Southern Blot, Hybridization, Produced, Clone Assay, Derivative Assay, FACS

ScPif1 and Rrm3 binding to centromeres is cell cycle regulated. Cells, which were grown at 24° throughout the experiment, were arrested by incubation in α-factor and then released to proceed through the cell cycle. Samples were collected for ChIP-qPCR and FACS at indicated times ( T = 0, 15, 30, 45, 60, 75, 90, and 105 min). Immunoprecipitated DNA was purified and analyzed by qPCR. Data are presented as [(ChIP/Input) Target site /(ChIP/Input) YBL028C ]. Error bars are 1 SD from the average value of three independent experiments. (A) Rrm3 binding to CEN3 throughout a synchronous cell cycle in WT cells (red circles) or in pif1-m2 cells (purple diamonds). (B) ScPif1 binding to CEN3 throughout a synchronous cell cycle in WT cells (blue squares) or in rrm3 Δ cells (green triangles). (C) Same as (A) except Rrm3 binding is to CEN11. (D) Same as in (B) except that ScPif1 binding is to CEN11. (E) Same as (A) except Rrm3 binding is to CEN12. (F) Same as in (B) except that ScPif1 binding is to CEN12. ChIP, chromatin immunoprecipitation; qPCR, quantitative PCR; WT, wild-type.
Figure Legend Snippet: ScPif1 and Rrm3 binding to centromeres is cell cycle regulated. Cells, which were grown at 24° throughout the experiment, were arrested by incubation in α-factor and then released to proceed through the cell cycle. Samples were collected for ChIP-qPCR and FACS at indicated times ( T = 0, 15, 30, 45, 60, 75, 90, and 105 min). Immunoprecipitated DNA was purified and analyzed by qPCR. Data are presented as [(ChIP/Input) Target site /(ChIP/Input) YBL028C ]. Error bars are 1 SD from the average value of three independent experiments. (A) Rrm3 binding to CEN3 throughout a synchronous cell cycle in WT cells (red circles) or in pif1-m2 cells (purple diamonds). (B) ScPif1 binding to CEN3 throughout a synchronous cell cycle in WT cells (blue squares) or in rrm3 Δ cells (green triangles). (C) Same as (A) except Rrm3 binding is to CEN11. (D) Same as in (B) except that ScPif1 binding is to CEN11. (E) Same as (A) except Rrm3 binding is to CEN12. (F) Same as in (B) except that ScPif1 binding is to CEN12. ChIP, chromatin immunoprecipitation; qPCR, quantitative PCR; WT, wild-type.

Techniques Used: Binding Assay, Incubation, Chromatin Immunoprecipitation, Real-time Polymerase Chain Reaction, FACS, Immunoprecipitation, Purification

34) Product Images from "Shallow whole genome sequencing for robust copy number profiling of formalin-fixed paraffin-embedded breast cancers"

Article Title: Shallow whole genome sequencing for robust copy number profiling of formalin-fixed paraffin-embedded breast cancers

Journal: Experimental and Molecular Pathology

doi: 10.1016/j.yexmp.2018.03.006

Features of input DNA and libraries generated from blocks less and more than five years. Dot plots represent the range (minimum-maximum) of observed values for each of the following categories and the red dot represents the median. A. The quality of input DNA inferred by ΔCt. B. Fragment sizes of the libraries in base pair. C. The library yield in nanomoles.
Figure Legend Snippet: Features of input DNA and libraries generated from blocks less and more than five years. Dot plots represent the range (minimum-maximum) of observed values for each of the following categories and the red dot represents the median. A. The quality of input DNA inferred by ΔCt. B. Fragment sizes of the libraries in base pair. C. The library yield in nanomoles.

Techniques Used: Generated

Features of the sequencing libraries. Boxplots showing different features of input DNA and library yield relative to the different library qualities. A. Input DNA. B. Quality of input DNA inferred from ΔCt. C. Fragment size of libraries. D. Library yield. VG = very good, G = good, I = intermediate, P = poor, F = fail.
Figure Legend Snippet: Features of the sequencing libraries. Boxplots showing different features of input DNA and library yield relative to the different library qualities. A. Input DNA. B. Quality of input DNA inferred from ΔCt. C. Fragment size of libraries. D. Library yield. VG = very good, G = good, I = intermediate, P = poor, F = fail.

Techniques Used: Sequencing

35) Product Images from "DNA methylation on N6-adenine in mammalian embryonic stem cells"

Article Title: DNA methylation on N6-adenine in mammalian embryonic stem cells

Journal: Nature

doi: 10.1038/nature17640

Alkbh1 is a specific N6-mA demethylase in vivo and in vitro a , Top: schematic of the CRISPR–Cas9 approach. Alkbh1 KO alleles don’t contain the XmaI site at exon 3. Bottom left: PCR-DNA digestion approach indicating the homozygosity of the knockout alleles, which are resistant to Xma1 digestion. Bottom right: western blotting did not detect any ALKBH1 proteins in the KO cells. b , Three additional Alkbh1 knockout ES cell clones show similar levels of N6-mA upregulation. Shown are dot blot results. c , Validating the specificity of anti-N6-mA antibodies with synthetic oligonucleotides. d , Validating the specificity of anti-N6-mA antibodies with DNA samples of different N6-mA/dA ratio. 125 ng of genomic DNA (MEFs) which does not contain any endogenous N6-mA was spiked with N6-mA containing oligonucleotides at the indicated concentration. e , Tandem mass spectrometric analysis shows the lack of H2AK118/119 methylation in wild-type or Alkbh1 knockout ES cells. Spectral counts for H2A peptides containing K118/119 revealed that H2AK118/119 is predominately non-methylated at similar levels between wild-type and Alkbh1 knockout ES cells. Spectral counts are reported as an average with standard deviation from biological triplicate analyses. K118/119: no methylation; K118/119me1: K118/119 monomethylation. f , MS analysis showed that the co-purified factors with recombinant ALKBH1 proteins are mainly heat shock proteins. g , ALKBH1 proteins don’t have noticeable activities towards to dual- or hemi-methylated double-stranded oligonucleotide substrates. h , ALKBH1 activities are dependent on Fe 2+ and α-KG. Error bars: standard deviation of triplicates. i , Ectopic expression of wild-type, but not mutant, Alkbh1 (D233A) at the catalytic motif, can rescue the aberrant increase of N6-mA level in Alkbh1 knockout ES cells. The wild-type and mutant Alkbh1 were expressed at similar levels. j , Quantification of three independent rescue experiments in i . P value as labelled, determined by t -test; error bars, s.d. for three biological replicates. k , The demethylation activity of N6-mA by recombinant D233A mutant protein is much reduced in comparison with the wild-type counterpart. l , No significant activities were detected with increasing concentrations of recombinant D233A mutant proteins in demethylation reaction. Error bars, s.d. of triplicates.
Figure Legend Snippet: Alkbh1 is a specific N6-mA demethylase in vivo and in vitro a , Top: schematic of the CRISPR–Cas9 approach. Alkbh1 KO alleles don’t contain the XmaI site at exon 3. Bottom left: PCR-DNA digestion approach indicating the homozygosity of the knockout alleles, which are resistant to Xma1 digestion. Bottom right: western blotting did not detect any ALKBH1 proteins in the KO cells. b , Three additional Alkbh1 knockout ES cell clones show similar levels of N6-mA upregulation. Shown are dot blot results. c , Validating the specificity of anti-N6-mA antibodies with synthetic oligonucleotides. d , Validating the specificity of anti-N6-mA antibodies with DNA samples of different N6-mA/dA ratio. 125 ng of genomic DNA (MEFs) which does not contain any endogenous N6-mA was spiked with N6-mA containing oligonucleotides at the indicated concentration. e , Tandem mass spectrometric analysis shows the lack of H2AK118/119 methylation in wild-type or Alkbh1 knockout ES cells. Spectral counts for H2A peptides containing K118/119 revealed that H2AK118/119 is predominately non-methylated at similar levels between wild-type and Alkbh1 knockout ES cells. Spectral counts are reported as an average with standard deviation from biological triplicate analyses. K118/119: no methylation; K118/119me1: K118/119 monomethylation. f , MS analysis showed that the co-purified factors with recombinant ALKBH1 proteins are mainly heat shock proteins. g , ALKBH1 proteins don’t have noticeable activities towards to dual- or hemi-methylated double-stranded oligonucleotide substrates. h , ALKBH1 activities are dependent on Fe 2+ and α-KG. Error bars: standard deviation of triplicates. i , Ectopic expression of wild-type, but not mutant, Alkbh1 (D233A) at the catalytic motif, can rescue the aberrant increase of N6-mA level in Alkbh1 knockout ES cells. The wild-type and mutant Alkbh1 were expressed at similar levels. j , Quantification of three independent rescue experiments in i . P value as labelled, determined by t -test; error bars, s.d. for three biological replicates. k , The demethylation activity of N6-mA by recombinant D233A mutant protein is much reduced in comparison with the wild-type counterpart. l , No significant activities were detected with increasing concentrations of recombinant D233A mutant proteins in demethylation reaction. Error bars, s.d. of triplicates.

Techniques Used: In Vivo, In Vitro, CRISPR, Polymerase Chain Reaction, Knock-Out, Western Blot, Clone Assay, Dot Blot, Concentration Assay, Methylation, Standard Deviation, Mass Spectrometry, Purification, Recombinant, Expressing, Mutagenesis, Activity Assay

36) Product Images from "Efficient yeast ChIP-Seq using multiplex short-read DNA sequencing"

Article Title: Efficient yeast ChIP-Seq using multiplex short-read DNA sequencing

Journal: BMC Genomics

doi: 10.1186/1471-2164-10-37

PolII signal profiles recapitulate findings from Steinmetz et al . PolII ChIP-Seq signal profiles resemble very closely to those published in Figure 3 of Steinmetz et al [ 54 ]. We obtained consistent binding at the Bap2-Tat1 loci (a) and at the Sed1-Shu2 loci (b). As expected, we did not observe binding at the Flo11 locus (c). For PolII ChIP-Seq experiments, two biological replicates were barcoded with ACGT (PolII_Rep1, dark blue; PolII_Rep2, orange), one was barcoded with TGCT (PolII_Rep3, red) and a fourth replicate had non-barcoded adapters (PolII_Rep4, green). Input DNA serves as a reference (light blue). Axis and scale normalizations are similar to Figure 2 . ORFs above the coordinates axis are on the Watson strand while ORFs below this axis are on the Crick strand.
Figure Legend Snippet: PolII signal profiles recapitulate findings from Steinmetz et al . PolII ChIP-Seq signal profiles resemble very closely to those published in Figure 3 of Steinmetz et al [ 54 ]. We obtained consistent binding at the Bap2-Tat1 loci (a) and at the Sed1-Shu2 loci (b). As expected, we did not observe binding at the Flo11 locus (c). For PolII ChIP-Seq experiments, two biological replicates were barcoded with ACGT (PolII_Rep1, dark blue; PolII_Rep2, orange), one was barcoded with TGCT (PolII_Rep3, red) and a fourth replicate had non-barcoded adapters (PolII_Rep4, green). Input DNA serves as a reference (light blue). Axis and scale normalizations are similar to Figure 2 . ORFs above the coordinates axis are on the Watson strand while ORFs below this axis are on the Crick strand.

Techniques Used: Chromatin Immunoprecipitation, Binding Assay

Comparison of input DNA signal tracks among all four barcoded adapters relative to standard Illumina adapters . An input sample was split in five aliquots. Four were barcoded differentially (top four lanes) and one had non-barcoded, Illumina adapters (fifth lane, labeled 'None'). Barcoded inputs were scored against non-barcoded input. IGB signal tracks of yeast chromosome 16 are shown for each sample, with ORF locations on the x-axis. ORFs are depicted in purple. On the y-axis, a normalized scale represents the number of read counts at a particular location. Each scale is normalized according to the number of mapped reads (Table 10 ). A box in the left panel depicts the enlarged section shown in the right panel for positions between 828,000 and 833,000 to demonstrate the overlap among all signal tracks.
Figure Legend Snippet: Comparison of input DNA signal tracks among all four barcoded adapters relative to standard Illumina adapters . An input sample was split in five aliquots. Four were barcoded differentially (top four lanes) and one had non-barcoded, Illumina adapters (fifth lane, labeled 'None'). Barcoded inputs were scored against non-barcoded input. IGB signal tracks of yeast chromosome 16 are shown for each sample, with ORF locations on the x-axis. ORFs are depicted in purple. On the y-axis, a normalized scale represents the number of read counts at a particular location. Each scale is normalized according to the number of mapped reads (Table 10 ). A box in the left panel depicts the enlarged section shown in the right panel for positions between 828,000 and 833,000 to demonstrate the overlap among all signal tracks.

Techniques Used: Labeling

Barcoded adapters perform similarly to standard Illumina adapters and do not crossover to other samples in the same lane . (a) RNA PolII binding profiles from different biological replicates with the same barcode (PolII_Rep1, dark blue; PolII_Rep3, red), with different barcodes (PolII_Rep2, orange) or without barcode (PolII_Rep4, green) strongly overlap. See also Table 3 . Input DNA serves as a reference (light blue). IGB signal tracks of chromosome 5 between 130,000 and 320,000 are shown for each library. A box in the left panel depicts the enlarged section shown in the right panel between positions 298,000 and 309,000 to illustrate the overlap among all PolII signal tracks. (b) Binding profiles from four different libraries pooled and sequenced in the same flowcell lane show very little resemblance. Shown here are the binding profiles for Cse4_Rep2 (dark blue), Ste12_Rep2 (red), PolII_Rep2 (green) and Input_ACGT (light blue). IGB signal tracks of chromosome 12 between 80,000 and 210,000 are shown for each sample. For (a) and (b), axis and scale normalizations are similar to Figure 2 . (c) Left: Rank-rank comparison of target lists between all pairwise barcoded replicates for Cse4, PolII and Ste12. The horizontal axis shows the fraction of the two lists being compared and the vertical axis shows the fraction of those targets that agree between a given pair of target lists. All comparisons show strong agreement, although the rank lists for Cse4 differ more than PolII or Ste12 for the second half of their length. Right: Rank-rank comparison between barcoded replicates from the same factors (averaged over all pairwise comparisons) compared to rank-rank comparisons for barcoded replicates between different factors: PolII_Rep1 (ACGT) vs. Ste12_Rep1 (TGCT) and Cse4_Rep2 (CATT) vs. Ste12_Rep2 (GTAT).
Figure Legend Snippet: Barcoded adapters perform similarly to standard Illumina adapters and do not crossover to other samples in the same lane . (a) RNA PolII binding profiles from different biological replicates with the same barcode (PolII_Rep1, dark blue; PolII_Rep3, red), with different barcodes (PolII_Rep2, orange) or without barcode (PolII_Rep4, green) strongly overlap. See also Table 3 . Input DNA serves as a reference (light blue). IGB signal tracks of chromosome 5 between 130,000 and 320,000 are shown for each library. A box in the left panel depicts the enlarged section shown in the right panel between positions 298,000 and 309,000 to illustrate the overlap among all PolII signal tracks. (b) Binding profiles from four different libraries pooled and sequenced in the same flowcell lane show very little resemblance. Shown here are the binding profiles for Cse4_Rep2 (dark blue), Ste12_Rep2 (red), PolII_Rep2 (green) and Input_ACGT (light blue). IGB signal tracks of chromosome 12 between 80,000 and 210,000 are shown for each sample. For (a) and (b), axis and scale normalizations are similar to Figure 2 . (c) Left: Rank-rank comparison of target lists between all pairwise barcoded replicates for Cse4, PolII and Ste12. The horizontal axis shows the fraction of the two lists being compared and the vertical axis shows the fraction of those targets that agree between a given pair of target lists. All comparisons show strong agreement, although the rank lists for Cse4 differ more than PolII or Ste12 for the second half of their length. Right: Rank-rank comparison between barcoded replicates from the same factors (averaged over all pairwise comparisons) compared to rank-rank comparisons for barcoded replicates between different factors: PolII_Rep1 (ACGT) vs. Ste12_Rep1 (TGCT) and Cse4_Rep2 (CATT) vs. Ste12_Rep2 (GTAT).

Techniques Used: Binding Assay

Ste12 distribution during pseudohyphal growth is similar across three different biological replicates . Two barcoded replicates (Ste12_Rep2, dark blue; Ste12_Rep1, red) and a non-barcoded replicate (Ste12_Rep3, green) were compared to input DNA (light blue). Ste12 ChIP samples were scored against a pool of input DNA. IGB signal tracks of chromosome 2 between 340,000 and 410,000 are shown for each sample. Axis and scale normalizations are similar to Figure 2 . A box in the left panel containing the TEC1 gene and its surrounding intergenic region was enlarged in panel B and rescaled to emphasize the strong signal at the TEC1 promoter. The same normalization as in Figure 2 was applied. Ste12p and Tec1p act as a dimer during pseudohyphal growth [ 31 ].
Figure Legend Snippet: Ste12 distribution during pseudohyphal growth is similar across three different biological replicates . Two barcoded replicates (Ste12_Rep2, dark blue; Ste12_Rep1, red) and a non-barcoded replicate (Ste12_Rep3, green) were compared to input DNA (light blue). Ste12 ChIP samples were scored against a pool of input DNA. IGB signal tracks of chromosome 2 between 340,000 and 410,000 are shown for each sample. Axis and scale normalizations are similar to Figure 2 . A box in the left panel containing the TEC1 gene and its surrounding intergenic region was enlarged in panel B and rescaled to emphasize the strong signal at the TEC1 promoter. The same normalization as in Figure 2 was applied. Ste12p and Tec1p act as a dimer during pseudohyphal growth [ 31 ].

Techniques Used: Chromatin Immunoprecipitation, Activated Clotting Time Assay

Cse4p is found robustly at centromeres . All biological replicates were strongly and tightly bound to centromeres, as it is depicted here in the case of CEN11 . Two barcoded replicates (Cse4_Rep2, dark blue; Cse4_Rep1, red) and a non-barcoded replicate (Cse4_Rep3, green) were compared to input DNA (light blue). Cse4 ChIP samples were scored against a pool of input DNA. IGB signal tracks of the CEN11 on chromosome 11 are shown for each sample. CEN11 is highlighted in a yellow box. Axis and scale normalizations are similar to Figure 2 .
Figure Legend Snippet: Cse4p is found robustly at centromeres . All biological replicates were strongly and tightly bound to centromeres, as it is depicted here in the case of CEN11 . Two barcoded replicates (Cse4_Rep2, dark blue; Cse4_Rep1, red) and a non-barcoded replicate (Cse4_Rep3, green) were compared to input DNA (light blue). Cse4 ChIP samples were scored against a pool of input DNA. IGB signal tracks of the CEN11 on chromosome 11 are shown for each sample. CEN11 is highlighted in a yellow box. Axis and scale normalizations are similar to Figure 2 .

Techniques Used: Chromatin Immunoprecipitation

Scheme for yeast barcoded ChIP-Seq . (a) Barcoded ChIP-Seq workflow. Ovals depict yeast cells and squares depict proteins. An aliquot of sheared cell lysate is not immunoprecipitated but is otherwise processed normally (green). This DNA, termed input DNA, is a reference sample for ChIP-Seq. Illumina DNA libraries are generated from both ChIP and input DNA samples. In multiplex ChIP-Seq, a barcoded adapter is ligated to an individual DNA sample. The barcode has 3 unique bases followed by a 'T' to anneal with the end-repaired DNA. Four libraries are then pooled together and applied to a single flowcell lane. After sequencing on the Genome Analyzer, reads are separated according to the first four bases and aligned to the yeast genome. Reads are stacked to generate a signal profile and scored against a pool of input DNA to determine significant transcription factor binding sites. (b) The barcode (orange) is located between Illumina adapter sequences (purple) and ChIP or input DNA inserts (black). The sequencing primer (pink) anneals to the adapter sequences and short reads start with the four bases of the barcode (orange) followed by DNA inserts (black). For the sequencing primer and Illumina adapter, oligonucleotide sequences were given by the manufacturer © 2006 Illumina, Inc. All rights reserved.
Figure Legend Snippet: Scheme for yeast barcoded ChIP-Seq . (a) Barcoded ChIP-Seq workflow. Ovals depict yeast cells and squares depict proteins. An aliquot of sheared cell lysate is not immunoprecipitated but is otherwise processed normally (green). This DNA, termed input DNA, is a reference sample for ChIP-Seq. Illumina DNA libraries are generated from both ChIP and input DNA samples. In multiplex ChIP-Seq, a barcoded adapter is ligated to an individual DNA sample. The barcode has 3 unique bases followed by a 'T' to anneal with the end-repaired DNA. Four libraries are then pooled together and applied to a single flowcell lane. After sequencing on the Genome Analyzer, reads are separated according to the first four bases and aligned to the yeast genome. Reads are stacked to generate a signal profile and scored against a pool of input DNA to determine significant transcription factor binding sites. (b) The barcode (orange) is located between Illumina adapter sequences (purple) and ChIP or input DNA inserts (black). The sequencing primer (pink) anneals to the adapter sequences and short reads start with the four bases of the barcode (orange) followed by DNA inserts (black). For the sequencing primer and Illumina adapter, oligonucleotide sequences were given by the manufacturer © 2006 Illumina, Inc. All rights reserved.

Techniques Used: Chromatin Immunoprecipitation, Immunoprecipitation, Generated, Multiplex Assay, Sequencing, Binding Assay

37) Product Images from "Pooled CRISPR Inverse PCR sequencing (PCIP-seq): simultaneous sequencing of retroviral insertion points and the integrated provirus with long reads"

Article Title: Pooled CRISPR Inverse PCR sequencing (PCIP-seq): simultaneous sequencing of retroviral insertion points and the integrated provirus with long reads

Journal: bioRxiv

doi: 10.1101/558130

PCIP-seq applied to ATL (a) In ATL100 both Illumina and Nanopore based methods show a single predominant insertion site (b) Screen shot from IGV shows a ∼16kb window with the provirus insertion site in the tumor clone identified via PCIP-seq and ligation mediated PCR with Illumina sequencing (c) PCIP-seq reads in IGV show a ∼3,600bp deletion in the provirus, confirmed via long range PCR and Illumina sequencing. (d) The ATL2 tumor clone contains three proviruses (named according to chromosome inserted into), the provirus on chr1 inserted into a repetitive element (LTR) and short reads generated from host DNA flanking the insertion site map to multiple positions in the genome. Filtering out multi-mapping reads causes an underestimation of the abundance of this insertion site (13.6 %), this can be partially corrected by retaining multi-mapping reads at this position (25.4 %). However, that approach can cause the potentially spurious inflation of other integration sites (red slice 9%). The long PCIP-seq reads can span repetitive elements and produce even coverage for each provirus without correction. (e) Screen shot from IGV shows representative reads coming from the three proviruses at positions where four de novo mutations were observed.
Figure Legend Snippet: PCIP-seq applied to ATL (a) In ATL100 both Illumina and Nanopore based methods show a single predominant insertion site (b) Screen shot from IGV shows a ∼16kb window with the provirus insertion site in the tumor clone identified via PCIP-seq and ligation mediated PCR with Illumina sequencing (c) PCIP-seq reads in IGV show a ∼3,600bp deletion in the provirus, confirmed via long range PCR and Illumina sequencing. (d) The ATL2 tumor clone contains three proviruses (named according to chromosome inserted into), the provirus on chr1 inserted into a repetitive element (LTR) and short reads generated from host DNA flanking the insertion site map to multiple positions in the genome. Filtering out multi-mapping reads causes an underestimation of the abundance of this insertion site (13.6 %), this can be partially corrected by retaining multi-mapping reads at this position (25.4 %). However, that approach can cause the potentially spurious inflation of other integration sites (red slice 9%). The long PCIP-seq reads can span repetitive elements and produce even coverage for each provirus without correction. (e) Screen shot from IGV shows representative reads coming from the three proviruses at positions where four de novo mutations were observed.

Techniques Used: Ligation, Polymerase Chain Reaction, Sequencing, Generated

38) Product Images from "Shallow whole genome sequencing for robust copy number profiling of formalin-fixed paraffin-embedded breast cancers"

Article Title: Shallow whole genome sequencing for robust copy number profiling of formalin-fixed paraffin-embedded breast cancers

Journal: Experimental and Molecular Pathology

doi: 10.1016/j.yexmp.2018.03.006

Features of the sequencing libraries. Boxplots showing different features of input DNA and library yield relative to the different library qualities. A. Input DNA. B. Quality of input DNA inferred from ΔCt. C. Fragment size of libraries. D. Library yield. VG = very good, G = good, I = intermediate, P = poor, F = fail.
Figure Legend Snippet: Features of the sequencing libraries. Boxplots showing different features of input DNA and library yield relative to the different library qualities. A. Input DNA. B. Quality of input DNA inferred from ΔCt. C. Fragment size of libraries. D. Library yield. VG = very good, G = good, I = intermediate, P = poor, F = fail.

Techniques Used: Sequencing

Features of input DNA and libraries generated from blocks less and more than five years. Dot plots represent the range (minimum-maximum) of observed values for each of the following categories and the red dot represents the median. A. The quality of input DNA inferred by ΔCt. B. Fragment sizes of the libraries in base pair. C. The library yield in nanomoles.
Figure Legend Snippet: Features of input DNA and libraries generated from blocks less and more than five years. Dot plots represent the range (minimum-maximum) of observed values for each of the following categories and the red dot represents the median. A. The quality of input DNA inferred by ΔCt. B. Fragment sizes of the libraries in base pair. C. The library yield in nanomoles.

Techniques Used: Generated

39) Product Images from "The long non‐coding RNA Paupar promotes KAP1‐dependent chromatin changes and regulates olfactory bulb neurogenesis"

Article Title: The long non‐coding RNA Paupar promotes KAP1‐dependent chromatin changes and regulates olfactory bulb neurogenesis

Journal: The EMBO Journal

doi: 10.15252/embj.201798219

Schematic detailing possible Paupar mode of action at distal bound regulatory regions Paupar promotes KAP1 chromatin association and H3K9me3 deposition through the assembly of a DNA bound ribonucleoprotein complex containing Paupar , KAP1 and PAX6 within the regulatory regions of direct target genes such as Mab21L2 , Mst1 and E2f2 . We propose three potential (non‐mutually exclusive) scenarios to describe the order of assembly of this complex: (i) A ternary complex forms in the nucleoplasm before binding DNA; (ii) Paupar interacts with KAP1 and guides it to DNA bound PAX6; or (iii) KAP1 is recruited to a DNA bound PAX6‐ Paupar complex. This leads to local H3K9me3 modification changes at these bound sequences in trans . The model was generated taking into consideration the discovery that Paupar genome‐wide binding sites contain an enrichment of motifs for neural transcription factors but are not enriched for sequences that are complementary to Paupar itself (Vance et al , 2014 ). This suggests that Paupar does not bind DNA directly but is targeted to chromatin indirectly through RNA–protein interactions with transcription factors such as PAX6. Moreover, KAP1 is a non‐DNA binding chromatin regulator that is also targeted to the genome through interactions with transcription factors.
Figure Legend Snippet: Schematic detailing possible Paupar mode of action at distal bound regulatory regions Paupar promotes KAP1 chromatin association and H3K9me3 deposition through the assembly of a DNA bound ribonucleoprotein complex containing Paupar , KAP1 and PAX6 within the regulatory regions of direct target genes such as Mab21L2 , Mst1 and E2f2 . We propose three potential (non‐mutually exclusive) scenarios to describe the order of assembly of this complex: (i) A ternary complex forms in the nucleoplasm before binding DNA; (ii) Paupar interacts with KAP1 and guides it to DNA bound PAX6; or (iii) KAP1 is recruited to a DNA bound PAX6‐ Paupar complex. This leads to local H3K9me3 modification changes at these bound sequences in trans . The model was generated taking into consideration the discovery that Paupar genome‐wide binding sites contain an enrichment of motifs for neural transcription factors but are not enriched for sequences that are complementary to Paupar itself (Vance et al , 2014 ). This suggests that Paupar does not bind DNA directly but is targeted to chromatin indirectly through RNA–protein interactions with transcription factors such as PAX6. Moreover, KAP1 is a non‐DNA binding chromatin regulator that is also targeted to the genome through interactions with transcription factors.

Techniques Used: Binding Assay, Modification, Generated, Genome Wide

Paupar co‐occupies a subset of KAP 1 binding sites on chromatin genome‐wide 5,510 KAP1 binding sites common to both replicates were identified relative to input DNA (1% FDR; Dataset EV4 ). Sites of KAP1 occupancy are particularly enriched at promoter regions (5′ UTRs), over gene bodies and over the 3′ UTR exons of zinc finger genes [ q = 2 × 10 −5 ; GAT randomisation test (Heger et al , 2013 )]. Intersection of KAP1 and Paupar binding sites in N2A cells identified 46 KAP1 bound locations that are specifically co‐occupied by Paupar . This represents a significant fourfold enrichment [ P
Figure Legend Snippet: Paupar co‐occupies a subset of KAP 1 binding sites on chromatin genome‐wide 5,510 KAP1 binding sites common to both replicates were identified relative to input DNA (1% FDR; Dataset EV4 ). Sites of KAP1 occupancy are particularly enriched at promoter regions (5′ UTRs), over gene bodies and over the 3′ UTR exons of zinc finger genes [ q = 2 × 10 −5 ; GAT randomisation test (Heger et al , 2013 )]. Intersection of KAP1 and Paupar binding sites in N2A cells identified 46 KAP1 bound locations that are specifically co‐occupied by Paupar . This represents a significant fourfold enrichment [ P

Techniques Used: Binding Assay, Genome Wide

Paupar promotes KAP 1 chromatin occupancy and H3K9me3 deposition at PAX 6 bound sequences within the regulatory regions of common targets Intersection of Paupar , KAP1 and PAX6 regulated genes identified 87 common target genes. 34 of these genes (in brackets) contain a Paupar binding site within their regulatory regions. ChIP assays were performed in N2A cells using either an antibody against KAP1 or an isotype‐specific control. N2A cells were transfected with either a non‐targeting control or two independent Paupar targeting shRNA expression vectors. Cells were harvested for ChIP 3 days later, and Paupar depletion was confirmed using qRT–PCR. Paupar knockdown reduces KAP1 chromatin occupancy at shared binding sites. ChIP assays were performed 3 days after shRNA transfection using an anti‐KAP1 polyclonal antibody. Western blotting showed that KAP1 proteins levels do not change upon Paupar knockdown. Actin was used as a control. Paupar promotes KAP1–PAX6 association. FLAG‐PAX6 and KAP1 expression vectors were co‐transfected into N2A cells along with increasing concentrations of Paupar or a size‐matched control lncRNA expression vector. Expression of the maximum concentration of either Paupar or control RNA in each IP does not alter KAP1 input protein levels (lower panel). Lysates were prepared 2 days after transfection and FLAG‐PAX6 protein immuno‐precipitated using anti‐FLAG beads. The amount of DNA transfected was made equal in each IP using empty vector and proteins in each complex were detected by Western blotting. Paupar knockdown reduces H3K9me3 at a subset of bound sequences in trans . ChIP assays were performed using an anti‐H3K9me3 polyclonal antibody 3 days after transfection of the indicated shRNA expression vectors. Data information: For ChIP assays, the indicated DNA fragments were amplified using qPCR. % input was calculated as 100 × 2 ( C t Input − C t IP ) . Results are presented as mean values ± SEM, N = 3. One‐tailed t ‐test, unequal variance * P
Figure Legend Snippet: Paupar promotes KAP 1 chromatin occupancy and H3K9me3 deposition at PAX 6 bound sequences within the regulatory regions of common targets Intersection of Paupar , KAP1 and PAX6 regulated genes identified 87 common target genes. 34 of these genes (in brackets) contain a Paupar binding site within their regulatory regions. ChIP assays were performed in N2A cells using either an antibody against KAP1 or an isotype‐specific control. N2A cells were transfected with either a non‐targeting control or two independent Paupar targeting shRNA expression vectors. Cells were harvested for ChIP 3 days later, and Paupar depletion was confirmed using qRT–PCR. Paupar knockdown reduces KAP1 chromatin occupancy at shared binding sites. ChIP assays were performed 3 days after shRNA transfection using an anti‐KAP1 polyclonal antibody. Western blotting showed that KAP1 proteins levels do not change upon Paupar knockdown. Actin was used as a control. Paupar promotes KAP1–PAX6 association. FLAG‐PAX6 and KAP1 expression vectors were co‐transfected into N2A cells along with increasing concentrations of Paupar or a size‐matched control lncRNA expression vector. Expression of the maximum concentration of either Paupar or control RNA in each IP does not alter KAP1 input protein levels (lower panel). Lysates were prepared 2 days after transfection and FLAG‐PAX6 protein immuno‐precipitated using anti‐FLAG beads. The amount of DNA transfected was made equal in each IP using empty vector and proteins in each complex were detected by Western blotting. Paupar knockdown reduces H3K9me3 at a subset of bound sequences in trans . ChIP assays were performed using an anti‐H3K9me3 polyclonal antibody 3 days after transfection of the indicated shRNA expression vectors. Data information: For ChIP assays, the indicated DNA fragments were amplified using qPCR. % input was calculated as 100 × 2 ( C t Input − C t IP ) . Results are presented as mean values ± SEM, N = 3. One‐tailed t ‐test, unequal variance * P

Techniques Used: Binding Assay, Chromatin Immunoprecipitation, Transfection, shRNA, Expressing, Quantitative RT-PCR, Western Blot, Plasmid Preparation, Concentration Assay, Amplification, Real-time Polymerase Chain Reaction, One-tailed Test

Related Articles

Methylation Sequencing:

Article Title: SINE transcription by RNA polymerase III is suppressed by histone methylation but not by DNA methylation
Article Snippet: .. ChIP-BS-Seq library preparation and sequencing For HeLa input DNA, two libraries were prepared from the same input DNA: one with starting quantity of 10 ng, following the Illumina ChIP-Seq library protocol, with the addition of bisulfite conversion after addition of adaptors but before amplification; and one following the Illumina Bisulfite Sequencing protocol, including the recommended starting amount. .. All libraries included addition of 2% sheared lambda DNA to control for bisulfite conversion.

Real-time Polymerase Chain Reaction:

Article Title: Activation of Oncogenic Super-Enhancers Is Coupled with DNA Repair by RAD51
Article Snippet: .. The ChIPed and the Input DNA were used for real-time PCR or to prepare libraries by Truseq (Ilumina) and sequenced in Nextseq (illumine). ..

Amplification:

Article Title: Impact of library preparation protocols and template quantity on the metagenomic reconstruction of a mock microbial community
Article Snippet: .. For example, work by Chafee et al. [ ] demonstrated that high quality metagenomic libraries can be constructed from as little as 50 pg of input DNA with the Nextera XT kit (Illumina), Solonenko et al. [ ] generated viral metagenomes with as little as 10 pg starting material using the Linear Amplification (LA) method, and Adey et al. [ ] demonstrated that the Nextera protocol can be used on just three copies of the human genome without significant coverage bias, the equivalent of 10 pg of genomic DNA. ..

Article Title: SINE transcription by RNA polymerase III is suppressed by histone methylation but not by DNA methylation
Article Snippet: .. ChIP-BS-Seq library preparation and sequencing For HeLa input DNA, two libraries were prepared from the same input DNA: one with starting quantity of 10 ng, following the Illumina ChIP-Seq library protocol, with the addition of bisulfite conversion after addition of adaptors but before amplification; and one following the Illumina Bisulfite Sequencing protocol, including the recommended starting amount. .. All libraries included addition of 2% sheared lambda DNA to control for bisulfite conversion.

Sequencing:

Article Title: Modulation of Enhancer Looping and Differential Gene Targeting by Epstein-Barr Virus Transcription Factors Directs Cellular Reprogramming
Article Snippet: .. Library preparation, sequencing and data analysis EBNA 2 ChIP and input DNA was used to generate sequencing libraries that were then subjected to 35 bp single-end read sequencing with an Illumina Genome Analyzer IIx as described previously . ..

Article Title: Rational “Error Elimination” Approach to Evaluating Molecular Barcoded Next-Generation Sequencing Data Identifies Low-Frequency Mutations in Hematologic Malignancies
Article Snippet: .. Typically, 50 ng of input DNA, 3 cycles of first-stage PCR with 0.5 μmol/L molecular barcode–containing 21-plex target-specific primer pairs, and 19 cycles of second-stage PCR with 0.5 μmol/L Illumina indexing primers yielded 1 to 10 nmol/L sequencing-ready libraries and produced molecular barcode families with a uniform size distribution. .. When the cutoff threshold for calling the variants from raw sequencing reads was reduced to 0.01%, > 50% of nucleotides in any given amplicon were found to yield low allele frequency variants (data not shown).

Article Title: SINE transcription by RNA polymerase III is suppressed by histone methylation but not by DNA methylation
Article Snippet: .. ChIP-BS-Seq library preparation and sequencing For HeLa input DNA, two libraries were prepared from the same input DNA: one with starting quantity of 10 ng, following the Illumina ChIP-Seq library protocol, with the addition of bisulfite conversion after addition of adaptors but before amplification; and one following the Illumina Bisulfite Sequencing protocol, including the recommended starting amount. .. All libraries included addition of 2% sheared lambda DNA to control for bisulfite conversion.

Construct:

Article Title: Impact of library preparation protocols and template quantity on the metagenomic reconstruction of a mock microbial community
Article Snippet: .. For example, work by Chafee et al. [ ] demonstrated that high quality metagenomic libraries can be constructed from as little as 50 pg of input DNA with the Nextera XT kit (Illumina), Solonenko et al. [ ] generated viral metagenomes with as little as 10 pg starting material using the Linear Amplification (LA) method, and Adey et al. [ ] demonstrated that the Nextera protocol can be used on just three copies of the human genome without significant coverage bias, the equivalent of 10 pg of genomic DNA. ..

Article Title: GT‐rich promoters can drive RNA pol II transcription and deposition of H2A.Z in African trypanosomes
Article Snippet: .. ChIP‐seq libraries were constructed using 35 ng of immunoprecipitated DNA or 35 ng of input DNA and sequenced on an Illumina® HiSeq 2500 or NextSeq 500. .. Mapping, normalization, and visualization of sequencing data Immunoprecipitated DNA samples were sequenced in paired‐end mode using an Illumina HiSeq 2500 or an Illumina NextSeq 500 sequencer with 2 × 100 and 2 × 76 cycles, respectively.

Produced:

Article Title: Rational “Error Elimination” Approach to Evaluating Molecular Barcoded Next-Generation Sequencing Data Identifies Low-Frequency Mutations in Hematologic Malignancies
Article Snippet: .. Typically, 50 ng of input DNA, 3 cycles of first-stage PCR with 0.5 μmol/L molecular barcode–containing 21-plex target-specific primer pairs, and 19 cycles of second-stage PCR with 0.5 μmol/L Illumina indexing primers yielded 1 to 10 nmol/L sequencing-ready libraries and produced molecular barcode families with a uniform size distribution. .. When the cutoff threshold for calling the variants from raw sequencing reads was reduced to 0.01%, > 50% of nucleotides in any given amplicon were found to yield low allele frequency variants (data not shown).

Immunoprecipitation:

Article Title: GT‐rich promoters can drive RNA pol II transcription and deposition of H2A.Z in African trypanosomes
Article Snippet: .. ChIP‐seq libraries were constructed using 35 ng of immunoprecipitated DNA or 35 ng of input DNA and sequenced on an Illumina® HiSeq 2500 or NextSeq 500. .. Mapping, normalization, and visualization of sequencing data Immunoprecipitated DNA samples were sequenced in paired‐end mode using an Illumina HiSeq 2500 or an Illumina NextSeq 500 sequencer with 2 × 100 and 2 × 76 cycles, respectively.

Generated:

Article Title: Impact of library preparation protocols and template quantity on the metagenomic reconstruction of a mock microbial community
Article Snippet: .. For example, work by Chafee et al. [ ] demonstrated that high quality metagenomic libraries can be constructed from as little as 50 pg of input DNA with the Nextera XT kit (Illumina), Solonenko et al. [ ] generated viral metagenomes with as little as 10 pg starting material using the Linear Amplification (LA) method, and Adey et al. [ ] demonstrated that the Nextera protocol can be used on just three copies of the human genome without significant coverage bias, the equivalent of 10 pg of genomic DNA. ..

Polymerase Chain Reaction:

Article Title: Transposon insertion libraries for the characterization of mutants from the kiwifruit pathogen Pseudomonas syringae pv. actinidiae
Article Snippet: .. An Illumina TruSeq Nano library was then created with ~1 μg input DNA using a Tru-Seq DNA Low-Throughput (LT) PCR-Free Library Kit (Illumina). .. Library DNA was resolved by gel electrophoresis in 6% w/v polyacrylamide, and a band of ~500 bp excised and purified.

Article Title: Quantitative BrdU immunoprecipitation method demonstrates that Fkh1 and Fkh2 are rate-limiting activators of replication origins that reprogram replication timing in G1 phase
Article Snippet: .. The IP and Input DNA samples were PCR-amplified separately with indexed Illumina-compatible primers. .. Amplified DNA was isolated using AMPure beads (Beckman Coulter, ) and validated and quantified on a Bioanalyzer (Agilent), and pooled.

Article Title: Rational “Error Elimination” Approach to Evaluating Molecular Barcoded Next-Generation Sequencing Data Identifies Low-Frequency Mutations in Hematologic Malignancies
Article Snippet: .. Typically, 50 ng of input DNA, 3 cycles of first-stage PCR with 0.5 μmol/L molecular barcode–containing 21-plex target-specific primer pairs, and 19 cycles of second-stage PCR with 0.5 μmol/L Illumina indexing primers yielded 1 to 10 nmol/L sequencing-ready libraries and produced molecular barcode families with a uniform size distribution. .. When the cutoff threshold for calling the variants from raw sequencing reads was reduced to 0.01%, > 50% of nucleotides in any given amplicon were found to yield low allele frequency variants (data not shown).

Chromatin Immunoprecipitation:

Article Title: Modulation of Enhancer Looping and Differential Gene Targeting by Epstein-Barr Virus Transcription Factors Directs Cellular Reprogramming
Article Snippet: .. Library preparation, sequencing and data analysis EBNA 2 ChIP and input DNA was used to generate sequencing libraries that were then subjected to 35 bp single-end read sequencing with an Illumina Genome Analyzer IIx as described previously . ..

Article Title: SINE transcription by RNA polymerase III is suppressed by histone methylation but not by DNA methylation
Article Snippet: .. ChIP-BS-Seq library preparation and sequencing For HeLa input DNA, two libraries were prepared from the same input DNA: one with starting quantity of 10 ng, following the Illumina ChIP-Seq library protocol, with the addition of bisulfite conversion after addition of adaptors but before amplification; and one following the Illumina Bisulfite Sequencing protocol, including the recommended starting amount. .. All libraries included addition of 2% sheared lambda DNA to control for bisulfite conversion.

ChIP-sequencing:

Article Title: GT‐rich promoters can drive RNA pol II transcription and deposition of H2A.Z in African trypanosomes
Article Snippet: .. ChIP‐seq libraries were constructed using 35 ng of immunoprecipitated DNA or 35 ng of input DNA and sequenced on an Illumina® HiSeq 2500 or NextSeq 500. .. Mapping, normalization, and visualization of sequencing data Immunoprecipitated DNA samples were sequenced in paired‐end mode using an Illumina HiSeq 2500 or an Illumina NextSeq 500 sequencer with 2 × 100 and 2 × 76 cycles, respectively.

Article Title: SINE transcription by RNA polymerase III is suppressed by histone methylation but not by DNA methylation
Article Snippet: .. ChIP-BS-Seq library preparation and sequencing For HeLa input DNA, two libraries were prepared from the same input DNA: one with starting quantity of 10 ng, following the Illumina ChIP-Seq library protocol, with the addition of bisulfite conversion after addition of adaptors but before amplification; and one following the Illumina Bisulfite Sequencing protocol, including the recommended starting amount. .. All libraries included addition of 2% sheared lambda DNA to control for bisulfite conversion.

Similar Products

  • Logo
  • About
  • News
  • Press Release
  • Team
  • Advisors
  • Partners
  • Contact
  • Bioz Stars
  • Bioz vStars
  • 88
    Illumina Inc chip input dna
    Genome-wide characterization of <t>Runx2</t> occupied regions (R2ORs). C4-2B/Rx2 dox cells were subjected to Runx2 ChIP-seq analysis after dox treatment and immunoprecipitation of Runx2-bound <t>DNA</t> fragments with anti-FLAG antibodies. ( A ) ChIP-seq results showing R2ORs upstream of the KLK and CSF2 TSS s . Raw ChIP-seq data are presented as frequency of reads per location for 30 kb of DNA sequences at the KLK2 (top) and CSF2 (bottom) loci. For each locus, the upper track shows results for chromatin aliquoted prior to immunoprecipitation (input). Regions investigated in Figure 1 by conventional ChIP assays are marked by black bars. ( B ) Sixteen Runx2 ChIP-seq peaks were validated by conventional ChIP-qPCR and the ChIP-seq scores are plotted against the qPCR enrichment factors. ( C ) Distance from the 1603 top R2ORs to the nearest TSS were mapped (red) and are shown along with control distribution patterns of 1000 sets of 1603 matched random sequences (gray). Y -axis values are numbers of R2ORs per 15 kb window. ( D ) Genomic distribution of the 1603 top R2ORs (red bars) versus that of 1000 sets of matched random sequences (gray bars; mean ± SD). TSS refers to −1000 to +100 bp from 5′-end of annotated RNA. Transcription Termination Site ( TTS ) is defined as −100 to +1000 bp from the 3′-end of the transcript. Inset shows blow up of TTS and TTS data.
    Chip Input Dna, supplied by Illumina Inc, used in various techniques. Bioz Stars score: 88/100, based on 6 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/chip input dna/product/Illumina Inc
    Average 88 stars, based on 6 article reviews
    Price from $9.99 to $1999.99
    chip input dna - by Bioz Stars, 2020-07
    88/100 stars
      Buy from Supplier

    89
    Illumina Inc hela input dna
    Pol III co-occupies methylated SINEs with MBPs. ( a ) Semiquantitative ChIP assay in A31 fibroblasts showing specific binding of TFIIIB, TFIIIC and pol III to B1 and B2 loci, as well as 7SL , but not the Apo-E gene. Histone H3 and TAF I 48 provide positive and negative controls, respectively. ( b ) Semiquantitative ChIP assay in <t>HeLa</t> cells showing occupancy of pol III, TFIIIB and TFIIIC at Alu loci from chromosomes 6, 10, 19 and 22, as well as 7SL and Apo-E genes. ChIPs for histone H3 and TAF I 48 provide positive and negative controls, respectively. No antibody was used for the mock sample. ( c ) Mean±s.e.m. of the percentage input bound in three independent ChIP–quantitative PCR (qPCR) assays in HeLa cells, of the indicated proteins at individual Alu loci from chromosomes 6, 10, 19 and 22, as well as 7SL and Apo-E loci and Alu PV subfamily consensus. ChIPs for histone H3 and TAF I 48 provide positive and negative controls, respectively. No antibody was used for the mock samples. P values are calculated by t -test. ( d ) Mean±s.e.m. of four independent sequential ChIP–qPCR assays in which <t>DNA</t> immunoprecipitated from HeLa cells using pol III antibody was reprecipitated using antibodies against pol III, TFIIIB, TAF I 48 (negative control), MBD1, MBD2 and MeCP2, as indicated. No TAF I 48 signal was detected on Alu(c6).
    Hela Input Dna, supplied by Illumina Inc, used in various techniques. Bioz Stars score: 89/100, based on 10 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/hela input dna/product/Illumina Inc
    Average 89 stars, based on 10 article reviews
    Price from $9.99 to $1999.99
    hela input dna - by Bioz Stars, 2020-07
    89/100 stars
      Buy from Supplier

    94
    Illumina Inc input dna
    Establishment of a high‐resolution MNase‐ChIP‐seq protocol for Trypanosoma brucei Outline of MNase‐ChIP‐seq. T. brucei cells were formaldehyde‐cross‐linked and permeabilized, and chromatin was digested into mononucleosomes using MNase. Nucleosomes containing histone H3 were isolated via affinity purification using rabbit H3 antiserum. After reversing cross‐links, the nucleosomal <t>DNA</t> was purified and paired‐end‐sequenced using <t>Illumina</t> HiSeq 2500. The sequencing reads were joined to fragments and assembled according to their midpoints. 2% agarose gel with 100 ng of mononucleosomal DNA after an MNase digest. Fragment size distribution after sequencing and joining of paired sequencing reads. Dashed lines indicate the fragment sizes 100, 137, 147, and 157 bp. Relative frequencies of AA/AT/TA/TT and CC/CG/GC/GG dinucleotides throughout 147 bp of nucleosomal DNA for each bp relative to the nucleosome dyad. Dashed lines indicate distance of 10 bp from position −74 bp.
    Input Dna, supplied by Illumina Inc, used in various techniques. Bioz Stars score: 94/100, based on 224 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/input dna/product/Illumina Inc
    Average 94 stars, based on 224 article reviews
    Price from $9.99 to $1999.99
    input dna - by Bioz Stars, 2020-07
    94/100 stars
      Buy from Supplier

    Image Search Results


    Genome-wide characterization of Runx2 occupied regions (R2ORs). C4-2B/Rx2 dox cells were subjected to Runx2 ChIP-seq analysis after dox treatment and immunoprecipitation of Runx2-bound DNA fragments with anti-FLAG antibodies. ( A ) ChIP-seq results showing R2ORs upstream of the KLK and CSF2 TSS s . Raw ChIP-seq data are presented as frequency of reads per location for 30 kb of DNA sequences at the KLK2 (top) and CSF2 (bottom) loci. For each locus, the upper track shows results for chromatin aliquoted prior to immunoprecipitation (input). Regions investigated in Figure 1 by conventional ChIP assays are marked by black bars. ( B ) Sixteen Runx2 ChIP-seq peaks were validated by conventional ChIP-qPCR and the ChIP-seq scores are plotted against the qPCR enrichment factors. ( C ) Distance from the 1603 top R2ORs to the nearest TSS were mapped (red) and are shown along with control distribution patterns of 1000 sets of 1603 matched random sequences (gray). Y -axis values are numbers of R2ORs per 15 kb window. ( D ) Genomic distribution of the 1603 top R2ORs (red bars) versus that of 1000 sets of matched random sequences (gray bars; mean ± SD). TSS refers to −1000 to +100 bp from 5′-end of annotated RNA. Transcription Termination Site ( TTS ) is defined as −100 to +1000 bp from the 3′-end of the transcript. Inset shows blow up of TTS and TTS data.

    Journal: Nucleic Acids Research

    Article Title: Genome-wide Runx2 occupancy in prostate cancer cells suggests a role in regulating secretion

    doi: 10.1093/nar/gkr1219

    Figure Lengend Snippet: Genome-wide characterization of Runx2 occupied regions (R2ORs). C4-2B/Rx2 dox cells were subjected to Runx2 ChIP-seq analysis after dox treatment and immunoprecipitation of Runx2-bound DNA fragments with anti-FLAG antibodies. ( A ) ChIP-seq results showing R2ORs upstream of the KLK and CSF2 TSS s . Raw ChIP-seq data are presented as frequency of reads per location for 30 kb of DNA sequences at the KLK2 (top) and CSF2 (bottom) loci. For each locus, the upper track shows results for chromatin aliquoted prior to immunoprecipitation (input). Regions investigated in Figure 1 by conventional ChIP assays are marked by black bars. ( B ) Sixteen Runx2 ChIP-seq peaks were validated by conventional ChIP-qPCR and the ChIP-seq scores are plotted against the qPCR enrichment factors. ( C ) Distance from the 1603 top R2ORs to the nearest TSS were mapped (red) and are shown along with control distribution patterns of 1000 sets of 1603 matched random sequences (gray). Y -axis values are numbers of R2ORs per 15 kb window. ( D ) Genomic distribution of the 1603 top R2ORs (red bars) versus that of 1000 sets of matched random sequences (gray bars; mean ± SD). TSS refers to −1000 to +100 bp from 5′-end of annotated RNA. Transcription Termination Site ( TTS ) is defined as −100 to +1000 bp from the 3′-end of the transcript. Inset shows blow up of TTS and TTS data.

    Article Snippet: ChIP-sequencing and peak calling Runx2 ChIP DNA along with ChIP input DNA were prepared as above from C4-2B/Rx2dox cells treated with dox for 14 h, and high throughput sequencing of the 500–1000 bp fragments was performed using Illumina Hi-Seq 2000.

    Techniques: Genome Wide, Chromatin Immunoprecipitation, Immunoprecipitation, Real-time Polymerase Chain Reaction

    DNA sequence motifs enriched in R2ORs. Motifs enriched in R2ORs compared to 1603 matched random sequences were identified using HOMER 3.1. ( A ) Logo for the top motif ( Runx2 ) is shown above the Runx1 logo identified by Pencovich et al. ( 35 ). ( B ) Motifs identified after R2ORs were re-analyzed as in A following masking of the Runx2 motifs. ( C ) Motif statistics. P- values are for motif enrichment. The percentages of R2ORs containing at least one copy of each motif are indicated against the percentage of motif-containing random sequences. Motifs/R2OR indicates for each motif the average number of copies per R2OR in R2ORs containing at least one copy.

    Journal: Nucleic Acids Research

    Article Title: Genome-wide Runx2 occupancy in prostate cancer cells suggests a role in regulating secretion

    doi: 10.1093/nar/gkr1219

    Figure Lengend Snippet: DNA sequence motifs enriched in R2ORs. Motifs enriched in R2ORs compared to 1603 matched random sequences were identified using HOMER 3.1. ( A ) Logo for the top motif ( Runx2 ) is shown above the Runx1 logo identified by Pencovich et al. ( 35 ). ( B ) Motifs identified after R2ORs were re-analyzed as in A following masking of the Runx2 motifs. ( C ) Motif statistics. P- values are for motif enrichment. The percentages of R2ORs containing at least one copy of each motif are indicated against the percentage of motif-containing random sequences. Motifs/R2OR indicates for each motif the average number of copies per R2OR in R2ORs containing at least one copy.

    Article Snippet: ChIP-sequencing and peak calling Runx2 ChIP DNA along with ChIP input DNA were prepared as above from C4-2B/Rx2dox cells treated with dox for 14 h, and high throughput sequencing of the 500–1000 bp fragments was performed using Illumina Hi-Seq 2000.

    Techniques: Sequencing

    Pol III co-occupies methylated SINEs with MBPs. ( a ) Semiquantitative ChIP assay in A31 fibroblasts showing specific binding of TFIIIB, TFIIIC and pol III to B1 and B2 loci, as well as 7SL , but not the Apo-E gene. Histone H3 and TAF I 48 provide positive and negative controls, respectively. ( b ) Semiquantitative ChIP assay in HeLa cells showing occupancy of pol III, TFIIIB and TFIIIC at Alu loci from chromosomes 6, 10, 19 and 22, as well as 7SL and Apo-E genes. ChIPs for histone H3 and TAF I 48 provide positive and negative controls, respectively. No antibody was used for the mock sample. ( c ) Mean±s.e.m. of the percentage input bound in three independent ChIP–quantitative PCR (qPCR) assays in HeLa cells, of the indicated proteins at individual Alu loci from chromosomes 6, 10, 19 and 22, as well as 7SL and Apo-E loci and Alu PV subfamily consensus. ChIPs for histone H3 and TAF I 48 provide positive and negative controls, respectively. No antibody was used for the mock samples. P values are calculated by t -test. ( d ) Mean±s.e.m. of four independent sequential ChIP–qPCR assays in which DNA immunoprecipitated from HeLa cells using pol III antibody was reprecipitated using antibodies against pol III, TFIIIB, TAF I 48 (negative control), MBD1, MBD2 and MeCP2, as indicated. No TAF I 48 signal was detected on Alu(c6).

    Journal: Nature Communications

    Article Title: SINE transcription by RNA polymerase III is suppressed by histone methylation but not by DNA methylation

    doi: 10.1038/ncomms7569

    Figure Lengend Snippet: Pol III co-occupies methylated SINEs with MBPs. ( a ) Semiquantitative ChIP assay in A31 fibroblasts showing specific binding of TFIIIB, TFIIIC and pol III to B1 and B2 loci, as well as 7SL , but not the Apo-E gene. Histone H3 and TAF I 48 provide positive and negative controls, respectively. ( b ) Semiquantitative ChIP assay in HeLa cells showing occupancy of pol III, TFIIIB and TFIIIC at Alu loci from chromosomes 6, 10, 19 and 22, as well as 7SL and Apo-E genes. ChIPs for histone H3 and TAF I 48 provide positive and negative controls, respectively. No antibody was used for the mock sample. ( c ) Mean±s.e.m. of the percentage input bound in three independent ChIP–quantitative PCR (qPCR) assays in HeLa cells, of the indicated proteins at individual Alu loci from chromosomes 6, 10, 19 and 22, as well as 7SL and Apo-E loci and Alu PV subfamily consensus. ChIPs for histone H3 and TAF I 48 provide positive and negative controls, respectively. No antibody was used for the mock samples. P values are calculated by t -test. ( d ) Mean±s.e.m. of four independent sequential ChIP–qPCR assays in which DNA immunoprecipitated from HeLa cells using pol III antibody was reprecipitated using antibodies against pol III, TFIIIB, TAF I 48 (negative control), MBD1, MBD2 and MeCP2, as indicated. No TAF I 48 signal was detected on Alu(c6).

    Article Snippet: ChIP-BS-Seq library preparation and sequencing For HeLa input DNA, two libraries were prepared from the same input DNA: one with starting quantity of 10 ng, following the Illumina ChIP-Seq library protocol, with the addition of bisulfite conversion after addition of adaptors but before amplification; and one following the Illumina Bisulfite Sequencing protocol, including the recommended starting amount.

    Techniques: Methylation, Chromatin Immunoprecipitation, Binding Assay, Real-time Polymerase Chain Reaction, Immunoprecipitation, Negative Control

    SINE expression is not stimulated by loss of DNA methylation. ( a ) Analysis by semiquantitative RT–PCR of expression levels of the indicated transcripts in matched Dnmt1 +/+ and Dnmt1 −/− fibroblasts. Duplicate samples are shown for both cell types. Apo-E and p53BP2 mRNAs provide controls that have been documented as being suppressed by DNA methylation. GAPDH mRNA provides a loading control. ( b ) Analysis by semiquantitative RT–PCR of expression levels of the indicated transcripts in mouse ES cells treated for 16 h with (+) or without (−) 5-azacytidine. Apo-E mRNA provides a control that has been documented as being inhibited by DNA methylation. ARPP P0 mRNA provides a loading control. ( c ) Analysis by semiquantitative RT–PCR of expression levels of the indicated transcripts in HeLa cells treated for 72 h with 5-azacytidine. Apo-E mRNA provides a control that has been documented as being inhibited by DNA methylation. ARPP P0 mRNA provides a loading control. ( d ) Analysis by primer extension of Alu transcripts in the RNA from Fig. 5c . Bracket indicates ~240 bp products that initiate at the principle pol III start site of Alu. Reverse transcriptase was omitted from the reactions in lanes 1 and 2. To confirm that the assay was not saturated, raising the amount of template RNA from 5 (lanes 5 and 6) to 10 μg (lanes 3 and 4) is shown to give a stronger signal. Alu, B1 and B2 RT–PCRs were performed with Alu, B1 and B2 consensus primers, respectively ( Supplementary Table 1 ).

    Journal: Nature Communications

    Article Title: SINE transcription by RNA polymerase III is suppressed by histone methylation but not by DNA methylation

    doi: 10.1038/ncomms7569

    Figure Lengend Snippet: SINE expression is not stimulated by loss of DNA methylation. ( a ) Analysis by semiquantitative RT–PCR of expression levels of the indicated transcripts in matched Dnmt1 +/+ and Dnmt1 −/− fibroblasts. Duplicate samples are shown for both cell types. Apo-E and p53BP2 mRNAs provide controls that have been documented as being suppressed by DNA methylation. GAPDH mRNA provides a loading control. ( b ) Analysis by semiquantitative RT–PCR of expression levels of the indicated transcripts in mouse ES cells treated for 16 h with (+) or without (−) 5-azacytidine. Apo-E mRNA provides a control that has been documented as being inhibited by DNA methylation. ARPP P0 mRNA provides a loading control. ( c ) Analysis by semiquantitative RT–PCR of expression levels of the indicated transcripts in HeLa cells treated for 72 h with 5-azacytidine. Apo-E mRNA provides a control that has been documented as being inhibited by DNA methylation. ARPP P0 mRNA provides a loading control. ( d ) Analysis by primer extension of Alu transcripts in the RNA from Fig. 5c . Bracket indicates ~240 bp products that initiate at the principle pol III start site of Alu. Reverse transcriptase was omitted from the reactions in lanes 1 and 2. To confirm that the assay was not saturated, raising the amount of template RNA from 5 (lanes 5 and 6) to 10 μg (lanes 3 and 4) is shown to give a stronger signal. Alu, B1 and B2 RT–PCRs were performed with Alu, B1 and B2 consensus primers, respectively ( Supplementary Table 1 ).

    Article Snippet: ChIP-BS-Seq library preparation and sequencing For HeLa input DNA, two libraries were prepared from the same input DNA: one with starting quantity of 10 ng, following the Illumina ChIP-Seq library protocol, with the addition of bisulfite conversion after addition of adaptors but before amplification; and one following the Illumina Bisulfite Sequencing protocol, including the recommended starting amount.

    Techniques: Expressing, DNA Methylation Assay, Reverse Transcription Polymerase Chain Reaction

    DNA methylation does not prevent pol III occupancy of SINEs. ( a ) Percentage input bound in three independent ChIP–quantitative PCR (qPCR) assays with mouse ES cells treated for 16 h with (+) or without (−) 5-azacytidine, showing occupancy of MBD2, MeCP2 and pol III at 7SL, B1 and B2 loci, as well as an Alu inserted onto chromosomes 14 and 17. ChIPs for TAF I 48 and without antibody (mock) provide negative controls. ( b ) Percentage input bound in three independent ChIP–qPCR assays with HeLa cells treated for 72 h with (+) or without (−) 5-azacytidine, showing the binding of MBD2, TFIIIB, TFIIIC and pol III to DNA centred over the body of Alu(c22) or 200 bp downstream. The resolution of this assay is limited by the size of the genomic DNA fragments (~500 bp). ( c ) Percentage input bound in two independent ChIP–qPCR assays with matched Dnmt1 +/+ and Dnmt1 −/− fibroblasts showing occupancy of MBD2, TFIIIB, TFIIIC and pol III at B1 and B2 loci, as well as 7SL and Apo-E genes. ChIPs for TAF I 48 and without antibody (mock) provide negative controls. Error bars indicate s.e.m. and all P values are calculated by t -test.

    Journal: Nature Communications

    Article Title: SINE transcription by RNA polymerase III is suppressed by histone methylation but not by DNA methylation

    doi: 10.1038/ncomms7569

    Figure Lengend Snippet: DNA methylation does not prevent pol III occupancy of SINEs. ( a ) Percentage input bound in three independent ChIP–quantitative PCR (qPCR) assays with mouse ES cells treated for 16 h with (+) or without (−) 5-azacytidine, showing occupancy of MBD2, MeCP2 and pol III at 7SL, B1 and B2 loci, as well as an Alu inserted onto chromosomes 14 and 17. ChIPs for TAF I 48 and without antibody (mock) provide negative controls. ( b ) Percentage input bound in three independent ChIP–qPCR assays with HeLa cells treated for 72 h with (+) or without (−) 5-azacytidine, showing the binding of MBD2, TFIIIB, TFIIIC and pol III to DNA centred over the body of Alu(c22) or 200 bp downstream. The resolution of this assay is limited by the size of the genomic DNA fragments (~500 bp). ( c ) Percentage input bound in two independent ChIP–qPCR assays with matched Dnmt1 +/+ and Dnmt1 −/− fibroblasts showing occupancy of MBD2, TFIIIB, TFIIIC and pol III at B1 and B2 loci, as well as 7SL and Apo-E genes. ChIPs for TAF I 48 and without antibody (mock) provide negative controls. Error bars indicate s.e.m. and all P values are calculated by t -test.

    Article Snippet: ChIP-BS-Seq library preparation and sequencing For HeLa input DNA, two libraries were prepared from the same input DNA: one with starting quantity of 10 ng, following the Illumina ChIP-Seq library protocol, with the addition of bisulfite conversion after addition of adaptors but before amplification; and one following the Illumina Bisulfite Sequencing protocol, including the recommended starting amount.

    Techniques: DNA Methylation Assay, Chromatin Immunoprecipitation, Real-time Polymerase Chain Reaction, Binding Assay

    Establishment of a high‐resolution MNase‐ChIP‐seq protocol for Trypanosoma brucei Outline of MNase‐ChIP‐seq. T. brucei cells were formaldehyde‐cross‐linked and permeabilized, and chromatin was digested into mononucleosomes using MNase. Nucleosomes containing histone H3 were isolated via affinity purification using rabbit H3 antiserum. After reversing cross‐links, the nucleosomal DNA was purified and paired‐end‐sequenced using Illumina HiSeq 2500. The sequencing reads were joined to fragments and assembled according to their midpoints. 2% agarose gel with 100 ng of mononucleosomal DNA after an MNase digest. Fragment size distribution after sequencing and joining of paired sequencing reads. Dashed lines indicate the fragment sizes 100, 137, 147, and 157 bp. Relative frequencies of AA/AT/TA/TT and CC/CG/GC/GG dinucleotides throughout 147 bp of nucleosomal DNA for each bp relative to the nucleosome dyad. Dashed lines indicate distance of 10 bp from position −74 bp.

    Journal: The EMBO Journal

    Article Title: GT‐rich promoters can drive RNA pol II transcription and deposition of H2A.Z in African trypanosomes

    doi: 10.15252/embj.201695323

    Figure Lengend Snippet: Establishment of a high‐resolution MNase‐ChIP‐seq protocol for Trypanosoma brucei Outline of MNase‐ChIP‐seq. T. brucei cells were formaldehyde‐cross‐linked and permeabilized, and chromatin was digested into mononucleosomes using MNase. Nucleosomes containing histone H3 were isolated via affinity purification using rabbit H3 antiserum. After reversing cross‐links, the nucleosomal DNA was purified and paired‐end‐sequenced using Illumina HiSeq 2500. The sequencing reads were joined to fragments and assembled according to their midpoints. 2% agarose gel with 100 ng of mononucleosomal DNA after an MNase digest. Fragment size distribution after sequencing and joining of paired sequencing reads. Dashed lines indicate the fragment sizes 100, 137, 147, and 157 bp. Relative frequencies of AA/AT/TA/TT and CC/CG/GC/GG dinucleotides throughout 147 bp of nucleosomal DNA for each bp relative to the nucleosome dyad. Dashed lines indicate distance of 10 bp from position −74 bp.

    Article Snippet: ChIP‐seq libraries were constructed using 35 ng of immunoprecipitated DNA or 35 ng of input DNA and sequenced on an Illumina® HiSeq 2500 or NextSeq 500.

    Techniques: Chromatin Immunoprecipitation, Isolation, Affinity Purification, Purification, Sequencing, Agarose Gel Electrophoresis

    Preliminary characterization of Psa transposon mutants. (A) Colony size variation between wild-type Psa (left) and transposon mutants (right). A region of each plate, boxed in black, is enlarged (2×) for comparison; (B) Evaluation of the ability of transposon mutants to express GUS on KB-Km agar medium containing X-Gluc; (C) Arbitrary PCR to amplify transposon insertion sites from the genomic DNA of 32 independent transposon mutants (1–32). PCR amplicons from samples labelled in red were sequenced to characterize the specific location(s) of genome insertion by the transposon. M = DNA ladder, bp = base pairs,– = H 2 O negative control, WT = wild-type Psa genomic DNA.

    Journal: PLoS ONE

    Article Title: Transposon insertion libraries for the characterization of mutants from the kiwifruit pathogen Pseudomonas syringae pv. actinidiae

    doi: 10.1371/journal.pone.0172790

    Figure Lengend Snippet: Preliminary characterization of Psa transposon mutants. (A) Colony size variation between wild-type Psa (left) and transposon mutants (right). A region of each plate, boxed in black, is enlarged (2×) for comparison; (B) Evaluation of the ability of transposon mutants to express GUS on KB-Km agar medium containing X-Gluc; (C) Arbitrary PCR to amplify transposon insertion sites from the genomic DNA of 32 independent transposon mutants (1–32). PCR amplicons from samples labelled in red were sequenced to characterize the specific location(s) of genome insertion by the transposon. M = DNA ladder, bp = base pairs,– = H 2 O negative control, WT = wild-type Psa genomic DNA.

    Article Snippet: An Illumina TruSeq Nano library was then created with ~1 μg input DNA using a Tru-Seq DNA Low-Throughput (LT) PCR-Free Library Kit (Illumina).

    Techniques: Polymerase Chain Reaction, Negative Control

    Validation of the Psa mutant of interest (MOI) library. (A) PCR screen to identify disruptions in the IYO_023025 gene. Pooled genomic DNA samples from the columns (lanes 1–12) and rows (lanes A–H) of MOI library plate 3 (P3) were used as templates for PCR. Two sets of amplicons that share a specific IYO_023025 disruption across a single pooled column and row sample are boxed in green and red, respectively; (B) Location of the P3-G10 and P3-D4 wells, which contain a mutant with an IYO_023025 disruption specific to the PCR amplicons boxed in green and red in (A), respectively; (C) Schematic of the IYO_023025 gene showing the location of transposon insertion sites identified in (A). Transposon insertion sites are denoted by arrows, and are color-coded to match the PCR amplicons boxed green and red in (A); (D) PCR screen to identify the IYO_023025 disruption mutant from well P3-G10. Pooled genomic DNA samples from the columns and rows of a 96-well plate (P2) containing independent colony-forming units of well P3-G10 were used as templates for PCR; (E) Location of intersecting wells in P2 (dark green) that possibly contain the IYO_023025 P3-G10 disruption mutant, as determined by the PCR amplicon profile in (D); (F) PCR screen to determine which of the intersecting wells in (E) contain the IYO_023025 P3-G10 disruption mutant. Amplicons shown in the left and right panels (separated by a black line) are derived from different regions of the same gel. Wells that contain the mutant are shown in bold in (E); (G) Colony morphology of wild-type (WT) Psa and the IYO_023025 P3-G10 disruption mutant. Bar = 2 μM, M = DNA ladder, bp = base pairs,– = H 2 O negative control, WT = WT Psa DNA.

    Journal: PLoS ONE

    Article Title: Transposon insertion libraries for the characterization of mutants from the kiwifruit pathogen Pseudomonas syringae pv. actinidiae

    doi: 10.1371/journal.pone.0172790

    Figure Lengend Snippet: Validation of the Psa mutant of interest (MOI) library. (A) PCR screen to identify disruptions in the IYO_023025 gene. Pooled genomic DNA samples from the columns (lanes 1–12) and rows (lanes A–H) of MOI library plate 3 (P3) were used as templates for PCR. Two sets of amplicons that share a specific IYO_023025 disruption across a single pooled column and row sample are boxed in green and red, respectively; (B) Location of the P3-G10 and P3-D4 wells, which contain a mutant with an IYO_023025 disruption specific to the PCR amplicons boxed in green and red in (A), respectively; (C) Schematic of the IYO_023025 gene showing the location of transposon insertion sites identified in (A). Transposon insertion sites are denoted by arrows, and are color-coded to match the PCR amplicons boxed green and red in (A); (D) PCR screen to identify the IYO_023025 disruption mutant from well P3-G10. Pooled genomic DNA samples from the columns and rows of a 96-well plate (P2) containing independent colony-forming units of well P3-G10 were used as templates for PCR; (E) Location of intersecting wells in P2 (dark green) that possibly contain the IYO_023025 P3-G10 disruption mutant, as determined by the PCR amplicon profile in (D); (F) PCR screen to determine which of the intersecting wells in (E) contain the IYO_023025 P3-G10 disruption mutant. Amplicons shown in the left and right panels (separated by a black line) are derived from different regions of the same gel. Wells that contain the mutant are shown in bold in (E); (G) Colony morphology of wild-type (WT) Psa and the IYO_023025 P3-G10 disruption mutant. Bar = 2 μM, M = DNA ladder, bp = base pairs,– = H 2 O negative control, WT = WT Psa DNA.

    Article Snippet: An Illumina TruSeq Nano library was then created with ~1 μg input DNA using a Tru-Seq DNA Low-Throughput (LT) PCR-Free Library Kit (Illumina).

    Techniques: Mutagenesis, Polymerase Chain Reaction, Amplification, Derivative Assay, Negative Control