Structured Review

Agilent technologies high sensitivity dna kit
Test case dataset samples ( a ) <t>Bioanalyzer</t> analysis to assess the quality of the starting material for Rep1, Rep2_bad and Rep2_good. Plots show the <t>DNA</t> size distribution after fragmentation. X-axis shows the fragment size in base pairs (bp) and y-axis the florescence intensity proportional to DNA abundance (FU = florescence unit). The target fragment length was about 200bp (vertical blue line), but “Rep2_bad” fragment size distribution is shifted to the left towards smaller sizes. ( b ) QC diagnostic plots based on EM and GM scores for the test case samples Rep1 (left column), Rep2_bad (centre column) and Rep2_good (right column). Cross correlation profiles (top rows), RSC score (red dashed line) plot against the reference distribution of RSC values (middle row) and fingerprint plots (bottom row) for the three test case samples. Overall, they do not consistently single out Rep2_bad as the real problematic sample.
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1) Product Images from "A ChIC solution for ChIP-seq quality assessment"

Article Title: A ChIC solution for ChIP-seq quality assessment

Journal: bioRxiv

doi: 10.1101/2020.05.19.103887

Test case dataset samples ( a ) Bioanalyzer analysis to assess the quality of the starting material for Rep1, Rep2_bad and Rep2_good. Plots show the DNA size distribution after fragmentation. X-axis shows the fragment size in base pairs (bp) and y-axis the florescence intensity proportional to DNA abundance (FU = florescence unit). The target fragment length was about 200bp (vertical blue line), but “Rep2_bad” fragment size distribution is shifted to the left towards smaller sizes. ( b ) QC diagnostic plots based on EM and GM scores for the test case samples Rep1 (left column), Rep2_bad (centre column) and Rep2_good (right column). Cross correlation profiles (top rows), RSC score (red dashed line) plot against the reference distribution of RSC values (middle row) and fingerprint plots (bottom row) for the three test case samples. Overall, they do not consistently single out Rep2_bad as the real problematic sample.
Figure Legend Snippet: Test case dataset samples ( a ) Bioanalyzer analysis to assess the quality of the starting material for Rep1, Rep2_bad and Rep2_good. Plots show the DNA size distribution after fragmentation. X-axis shows the fragment size in base pairs (bp) and y-axis the florescence intensity proportional to DNA abundance (FU = florescence unit). The target fragment length was about 200bp (vertical blue line), but “Rep2_bad” fragment size distribution is shifted to the left towards smaller sizes. ( b ) QC diagnostic plots based on EM and GM scores for the test case samples Rep1 (left column), Rep2_bad (centre column) and Rep2_good (right column). Cross correlation profiles (top rows), RSC score (red dashed line) plot against the reference distribution of RSC values (middle row) and fingerprint plots (bottom row) for the three test case samples. Overall, they do not consistently single out Rep2_bad as the real problematic sample.

Techniques Used: Diagnostic Assay

2) Product Images from "Mapping chromatin accessibility and active regulatory elements reveals new pathological mechanisms in human gliomas"

Article Title: Mapping chromatin accessibility and active regulatory elements reveals new pathological mechanisms in human gliomas

Journal: bioRxiv

doi: 10.1101/867861

A. A characteristic appearance of a single nucleosome and their multiplications in the material isolated for ATAC-seq separated on DNA Agilent chips (PA04 sample). B. Validation of immunoprecipitation efficiency with antibodies used in the experiments (for details see Methods). Chromatin immunoprecipitation (ChIP)-quantitative polymerase chain reaction (qPCR) analysis of histone modification enrichment in control regions: GAPDH promoter (active chromatin marks, H3K4me3, H3K27ac) and HOXA7 gene body (repressive mark, H3K27me3). Results are calculated as fold enrichment over negative control (immunoprecipitation with normal IgG) and represented as mean ± SD (H3K4me3 ChIP-qPCR n=6, H3K27ac ChIP-qPCR n=5, H3K27me3 ChIP-qPCR n=3n = 4). Statistical significance **P
Figure Legend Snippet: A. A characteristic appearance of a single nucleosome and their multiplications in the material isolated for ATAC-seq separated on DNA Agilent chips (PA04 sample). B. Validation of immunoprecipitation efficiency with antibodies used in the experiments (for details see Methods). Chromatin immunoprecipitation (ChIP)-quantitative polymerase chain reaction (qPCR) analysis of histone modification enrichment in control regions: GAPDH promoter (active chromatin marks, H3K4me3, H3K27ac) and HOXA7 gene body (repressive mark, H3K27me3). Results are calculated as fold enrichment over negative control (immunoprecipitation with normal IgG) and represented as mean ± SD (H3K4me3 ChIP-qPCR n=6, H3K27ac ChIP-qPCR n=5, H3K27me3 ChIP-qPCR n=3n = 4). Statistical significance **P

Techniques Used: Isolation, Immunoprecipitation, Chromatin Immunoprecipitation, Real-time Polymerase Chain Reaction, Modification, Negative Control

3) Product Images from "Expanded subgenomic mRNA transcriptome and coding capacity of a nidovirus"

Article Title: Expanded subgenomic mRNA transcriptome and coding capacity of a nidovirus

Journal: Proceedings of the National Academy of Sciences of the United States of America

doi: 10.1073/pnas.1706696114

Amplification and cloning of sg mRNA leader–body junctions generated from additional functional TRSs located in the genomic region between identified TRS5 and TRS6. ( A ) Diagram indicating the positions of the primers used and the estimated size for the amplified leader–body junction sequences (thick black line). The white open box represents ORF5, and the short black box represents the leader sequence in transcribed sg mRNAs. ( B ) MA104 cells were either mock-infected (M) or infected with SHFVic at an MOI of 1. At 24 hpi, total intracellular RNA was extracted and subjected to RT-PCR, and the products were separated on a 2% DNA gel. The band with the size estimated for the leader–body junction in sg mRNA5 produced from the known TRS5 is indicated by an arrow. PCR bands with sizes estimated for the leader–body junctions of ∼1.7-kb sg mRNAs are indicated by a bracket. L, ladder. ( C ) The bracketed region of the gel was excised, and the DNA was extracted and cloned into a TA vector. Forty colonies were randomly selected and subjected to restriction digestion, and the inserts were separated by gel electrophoresis. The results from 10 representative clones are shown. L, ladder. ( D ) Diagram showing the locations of the known and previously unreported body TRSs. The TRSs are indicated by black vertical bars. The previously unreported functional TRSs are within a dotted line box. The ORFs encoded by the individual sg mRNAs are indicated by white open boxes. 5-C, ORF5-C-68aa; L, leader region.
Figure Legend Snippet: Amplification and cloning of sg mRNA leader–body junctions generated from additional functional TRSs located in the genomic region between identified TRS5 and TRS6. ( A ) Diagram indicating the positions of the primers used and the estimated size for the amplified leader–body junction sequences (thick black line). The white open box represents ORF5, and the short black box represents the leader sequence in transcribed sg mRNAs. ( B ) MA104 cells were either mock-infected (M) or infected with SHFVic at an MOI of 1. At 24 hpi, total intracellular RNA was extracted and subjected to RT-PCR, and the products were separated on a 2% DNA gel. The band with the size estimated for the leader–body junction in sg mRNA5 produced from the known TRS5 is indicated by an arrow. PCR bands with sizes estimated for the leader–body junctions of ∼1.7-kb sg mRNAs are indicated by a bracket. L, ladder. ( C ) The bracketed region of the gel was excised, and the DNA was extracted and cloned into a TA vector. Forty colonies were randomly selected and subjected to restriction digestion, and the inserts were separated by gel electrophoresis. The results from 10 representative clones are shown. L, ladder. ( D ) Diagram showing the locations of the known and previously unreported body TRSs. The TRSs are indicated by black vertical bars. The previously unreported functional TRSs are within a dotted line box. The ORFs encoded by the individual sg mRNAs are indicated by white open boxes. 5-C, ORF5-C-68aa; L, leader region.

Techniques Used: Amplification, Clone Assay, Generated, Functional Assay, Sequencing, Infection, Reverse Transcription Polymerase Chain Reaction, Produced, Polymerase Chain Reaction, Plasmid Preparation, Nucleic Acid Electrophoresis

4) Product Images from "Expanding Duplication of Free Fatty Acid Receptor-2 (GPR43) Genes in the Chicken Genome"

Article Title: Expanding Duplication of Free Fatty Acid Receptor-2 (GPR43) Genes in the Chicken Genome

Journal: Genome Biology and Evolution

doi: 10.1093/gbe/evv072

FFAR2 has numerous copies in European broiler and in ancestral chicken genome. qPCR on genomic DNA shows clear difference in ΔCq between FFAR2 genes and the genes carrying only one copy per genome (four genes in European lines and three genes in Ancestral lines). qPCR was performed using “universal” primers able to amplify the 26 sequences of FFAR2 (see supplementary data S1 , Supplementary Material online). FFAR2 error bars indicate standard deviation between two individual chickens. For the single copy genes, error bars represent standard deviation between the Cq measures for the three or four genes from two individual chickens.
Figure Legend Snippet: FFAR2 has numerous copies in European broiler and in ancestral chicken genome. qPCR on genomic DNA shows clear difference in ΔCq between FFAR2 genes and the genes carrying only one copy per genome (four genes in European lines and three genes in Ancestral lines). qPCR was performed using “universal” primers able to amplify the 26 sequences of FFAR2 (see supplementary data S1 , Supplementary Material online). FFAR2 error bars indicate standard deviation between two individual chickens. For the single copy genes, error bars represent standard deviation between the Cq measures for the three or four genes from two individual chickens.

Techniques Used: Real-time Polymerase Chain Reaction, Standard Deviation

5) Product Images from "Indication of Horizontal DNA Gene Transfer by Extracellular Vesicles"

Article Title: Indication of Horizontal DNA Gene Transfer by Extracellular Vesicles

Journal: PLoS ONE

doi: 10.1371/journal.pone.0163665

Detection of DNA in extracellular vesicles. EV were isolated from supernatants of hMSC by ultracentrifugation, divided into two parts and DNA prepared from EV without DNase treatment (a) or after DNase treatment (b). For workflow see S4 Fig . Automatically set standards of 35 (green) and 10380 bp (pink) in the Bioanalyzer indicate the lower and upper size markers. Shown are the Bioanalyzer profiles and respective gels for a representative example. (c) Ten μl from a total of 40 μl DNA sample isolated from EV without (EV no DNase) or with (EV + DNase) DNase treatment were separated on a 0.66% agarose gel. (d) To analyze the localization, DNA was isolated from unmanipulated EV (-DNase) or EV after DNase treatment (+DNase) and examined for genomic signals in quantitative PCR using primer pairs for GAPDH, BC32-A and BD16-C1 (both randomly chosen from human genome sequences). Shown are the mean Ct values ± SD of two experiments carried out in duplicates (left graph). Products of one experiment in duplicates were visualized on 1.8% agarose gels (right blots). NTC: no template control. (e) To further elucidate the composition of EV in regard to their DNA cargo, 10 μg of protein lysate were separated on 15% SDS-PAGE and analyzed for histones H1, H2B, H3, H4 and GAPDH. As positive control, a nuclear fraction (nf) of human H1299 cells was used.
Figure Legend Snippet: Detection of DNA in extracellular vesicles. EV were isolated from supernatants of hMSC by ultracentrifugation, divided into two parts and DNA prepared from EV without DNase treatment (a) or after DNase treatment (b). For workflow see S4 Fig . Automatically set standards of 35 (green) and 10380 bp (pink) in the Bioanalyzer indicate the lower and upper size markers. Shown are the Bioanalyzer profiles and respective gels for a representative example. (c) Ten μl from a total of 40 μl DNA sample isolated from EV without (EV no DNase) or with (EV + DNase) DNase treatment were separated on a 0.66% agarose gel. (d) To analyze the localization, DNA was isolated from unmanipulated EV (-DNase) or EV after DNase treatment (+DNase) and examined for genomic signals in quantitative PCR using primer pairs for GAPDH, BC32-A and BD16-C1 (both randomly chosen from human genome sequences). Shown are the mean Ct values ± SD of two experiments carried out in duplicates (left graph). Products of one experiment in duplicates were visualized on 1.8% agarose gels (right blots). NTC: no template control. (e) To further elucidate the composition of EV in regard to their DNA cargo, 10 μg of protein lysate were separated on 15% SDS-PAGE and analyzed for histones H1, H2B, H3, H4 and GAPDH. As positive control, a nuclear fraction (nf) of human H1299 cells was used.

Techniques Used: Isolation, Agarose Gel Electrophoresis, Real-time Polymerase Chain Reaction, SDS Page, Positive Control

6) Product Images from "Quartz-Seq2: a high-throughput single-cell RNA-sequencing method that effectively uses limited sequence reads"

Article Title: Quartz-Seq2: a high-throughput single-cell RNA-sequencing method that effectively uses limited sequence reads

Journal: Genome Biology

doi: 10.1186/s13059-018-1407-3

Overview of Quartz-Seq2 experimental processes. a Quartz-Seq2 consists of five steps. (1) Each single cell in a droplet is sorted into lysis buffer in each well of a 384-well PCR plate using flow cytometry analysis data. (2) Poly-adenylated RNA in each well is reverse-transcribed into first-strand cDNA with reverse transcription primer, which has a unique cell barcode ( CB ). We prepare 384 or 1536 kinds of cell barcode with a unique sequence based on the Sequence–Levenshtein distance (SeqLv). The edit distance of SeqLv is 5. The RT primer also has a UMI sequence for reduction of PCR bias (MB) and a poly(dT) sequence for binding to poly(A) RNA. (3) Cell barcode-labeled cDNAs from all 384 wells are promptly collected by centrifugation using assembled collectors. (4) Collected first-strand cDNAs are purified and concentrated for subsequent whole-transcript amplification. In the poly(A) tailing step, purified cDNA is extended with a poly(A) tail by terminal deoxynucleotidyl transferase ( TdT ). Subsequently, second-strand cDNA is synthesized with a tagging primer, which has a poly(dT) sequence. The resulting second-strand cDNA has a PCR primer sequence ( M ) at both ends of it. The cDNA is amplifiable in a subsequent PCR amplification. (5) For conversion from amplified cDNA to sequence library DNA, we fragment the amplified cDNA using the ultrasonicator Covaris. Such fragmented cDNA is ligated with a truncated Y-shaped sequence adaptor, which has an Illumina flow-cell binding sequence ( P7 ) and a pool barcode sequence ( PB ). The PB makes it possible to mix different sets of cell barcode-labeled cDNA. Ligated cDNA, which has CB and MB sequences, is enriched by PCR amplification. The resulting sequence library DNA contains P7 and P5 flow-cell binding sequences at respective ends of the DNA. We sequence the cell barcode site and the UMI site at Read1, the pool barcode site at Index1, and the transcript sequence at Read2. b The relationship between initial fastq reads and the number of single cells for sequence analysis in NextSeq500 runs. Typically, one sequence run with NextSeq 500/550 High Output v2 Kit reads out 400–450 M fastq reads. The x-axis represents the input cell number for one sequence run. The y-axis represents the initial data size (fastq reads) on average per cell. The red outline represents the typical range of shallow input read depth for a single cell. c We define the formula for calculating the UMI conversion efficiency. Each parameter is defined as follows: UMI sc is the number of UMI counts, assigned to a single-cell sample, fastq sc is the number of fastq reads derived from each single-cell sample, fastq non-sc is the number of fastq reads derived from non-single-cell samples, which include experimental byproducts such as WTA adaptors, WTA byproducts, and non-STAMPs. Initial fastq reads are composed of fastq sc and fastq non-sc
Figure Legend Snippet: Overview of Quartz-Seq2 experimental processes. a Quartz-Seq2 consists of five steps. (1) Each single cell in a droplet is sorted into lysis buffer in each well of a 384-well PCR plate using flow cytometry analysis data. (2) Poly-adenylated RNA in each well is reverse-transcribed into first-strand cDNA with reverse transcription primer, which has a unique cell barcode ( CB ). We prepare 384 or 1536 kinds of cell barcode with a unique sequence based on the Sequence–Levenshtein distance (SeqLv). The edit distance of SeqLv is 5. The RT primer also has a UMI sequence for reduction of PCR bias (MB) and a poly(dT) sequence for binding to poly(A) RNA. (3) Cell barcode-labeled cDNAs from all 384 wells are promptly collected by centrifugation using assembled collectors. (4) Collected first-strand cDNAs are purified and concentrated for subsequent whole-transcript amplification. In the poly(A) tailing step, purified cDNA is extended with a poly(A) tail by terminal deoxynucleotidyl transferase ( TdT ). Subsequently, second-strand cDNA is synthesized with a tagging primer, which has a poly(dT) sequence. The resulting second-strand cDNA has a PCR primer sequence ( M ) at both ends of it. The cDNA is amplifiable in a subsequent PCR amplification. (5) For conversion from amplified cDNA to sequence library DNA, we fragment the amplified cDNA using the ultrasonicator Covaris. Such fragmented cDNA is ligated with a truncated Y-shaped sequence adaptor, which has an Illumina flow-cell binding sequence ( P7 ) and a pool barcode sequence ( PB ). The PB makes it possible to mix different sets of cell barcode-labeled cDNA. Ligated cDNA, which has CB and MB sequences, is enriched by PCR amplification. The resulting sequence library DNA contains P7 and P5 flow-cell binding sequences at respective ends of the DNA. We sequence the cell barcode site and the UMI site at Read1, the pool barcode site at Index1, and the transcript sequence at Read2. b The relationship between initial fastq reads and the number of single cells for sequence analysis in NextSeq500 runs. Typically, one sequence run with NextSeq 500/550 High Output v2 Kit reads out 400–450 M fastq reads. The x-axis represents the input cell number for one sequence run. The y-axis represents the initial data size (fastq reads) on average per cell. The red outline represents the typical range of shallow input read depth for a single cell. c We define the formula for calculating the UMI conversion efficiency. Each parameter is defined as follows: UMI sc is the number of UMI counts, assigned to a single-cell sample, fastq sc is the number of fastq reads derived from each single-cell sample, fastq non-sc is the number of fastq reads derived from non-single-cell samples, which include experimental byproducts such as WTA adaptors, WTA byproducts, and non-STAMPs. Initial fastq reads are composed of fastq sc and fastq non-sc

Techniques Used: Lysis, Polymerase Chain Reaction, Flow Cytometry, Cytometry, Sequencing, Binding Assay, Labeling, Centrifugation, Purification, Amplification, Synthesized, Derivative Assay

Sequence performance of Quartz-Seq2 with molecular biological improvements. a Improvement of poly(A) tagging efficiency. The relative DNA yield in various poly(A) tagging conditions using purified first-strand cDNA from 1 ng of total RNA. T55 buffer as the terminal deoxynucleotidyl transferase (TdT) buffer and the temperature condition “ Increment ” for the poly(A) tagging step improved the cDNA yield of whole-transcript amplification. Buffer compositions are indicated in Additional file 6 : Table S5. QuartzB represents use of a Quartz-Seq-like buffer as a positive control, in accordance with the approach described in the original Quartz-Seq paper. Finally, we quantified cDNA yield (300–9000 bp) and byproduct DNA yield (50–300 bp) using a Bioanalyzer (Agilent). The presented p value was obtained using two-tailed Welch’s t -test. b Reverse transcription efficiency with serially diluted RT enzymes. The x-axis represents the average relative RT qPCR score from ten genes. Detailed concentrations of RT enzymes are presented in Additional file 1 : Figure S7. c , f – h Comparison between Quartz-Seq2 in the RT25 condition and Quartz-Seq-like conditions regarding sequence performance. c We analyzed 384 wells with 10 pg of total RNA and used approximately 0.19 M fastq reads on average per well. We show the UMI count and gene count in box plots. d A scatter plot between the mean of gene expression and the variability of gene expression with 10 pg of total RNA in 384 wells. Red lines represent the theoretical variability of gene expression in the form of a Poisson distribution. e Gene expression reproducibility between bulk poly(A)-RNA-seq (1 μg of total RNA) and Quartz-Seq2 (10 pg of total RNA, averaged over 384 wells). f Dispersion of gene expression. The x-axis represents gene expression variability. g Reproducibility of gene expression for internal gene and external control RNA. h Accuracy of gene expression for internal gene and external control RNA
Figure Legend Snippet: Sequence performance of Quartz-Seq2 with molecular biological improvements. a Improvement of poly(A) tagging efficiency. The relative DNA yield in various poly(A) tagging conditions using purified first-strand cDNA from 1 ng of total RNA. T55 buffer as the terminal deoxynucleotidyl transferase (TdT) buffer and the temperature condition “ Increment ” for the poly(A) tagging step improved the cDNA yield of whole-transcript amplification. Buffer compositions are indicated in Additional file 6 : Table S5. QuartzB represents use of a Quartz-Seq-like buffer as a positive control, in accordance with the approach described in the original Quartz-Seq paper. Finally, we quantified cDNA yield (300–9000 bp) and byproduct DNA yield (50–300 bp) using a Bioanalyzer (Agilent). The presented p value was obtained using two-tailed Welch’s t -test. b Reverse transcription efficiency with serially diluted RT enzymes. The x-axis represents the average relative RT qPCR score from ten genes. Detailed concentrations of RT enzymes are presented in Additional file 1 : Figure S7. c , f – h Comparison between Quartz-Seq2 in the RT25 condition and Quartz-Seq-like conditions regarding sequence performance. c We analyzed 384 wells with 10 pg of total RNA and used approximately 0.19 M fastq reads on average per well. We show the UMI count and gene count in box plots. d A scatter plot between the mean of gene expression and the variability of gene expression with 10 pg of total RNA in 384 wells. Red lines represent the theoretical variability of gene expression in the form of a Poisson distribution. e Gene expression reproducibility between bulk poly(A)-RNA-seq (1 μg of total RNA) and Quartz-Seq2 (10 pg of total RNA, averaged over 384 wells). f Dispersion of gene expression. The x-axis represents gene expression variability. g Reproducibility of gene expression for internal gene and external control RNA. h Accuracy of gene expression for internal gene and external control RNA

Techniques Used: Sequencing, Purification, Amplification, Positive Control, Two Tailed Test, Quantitative RT-PCR, Expressing, RNA Sequencing Assay

7) Product Images from "Optimization of Extraction of Circulating RNAs from Plasma – Enabling Small RNA Sequencing"

Article Title: Optimization of Extraction of Circulating RNAs from Plasma – Enabling Small RNA Sequencing

Journal: PLoS ONE

doi: 10.1371/journal.pone.0107259

High Sensitivity DNA Bioanalyzer assay as checkpoint for correct size selection during library preparation. All nine samples showed adaptor/RNA/adaptor-constructs in appropriate sizes. One electropherogram is shown as representative example. The lengths of adaptor-ligated constructs from all nine samples were reported as indicated in the column peak size [bp] . The initial peak at 35 bp and the final peak at 10.380 bp are marker peaks that are system inherent included in all runs.
Figure Legend Snippet: High Sensitivity DNA Bioanalyzer assay as checkpoint for correct size selection during library preparation. All nine samples showed adaptor/RNA/adaptor-constructs in appropriate sizes. One electropherogram is shown as representative example. The lengths of adaptor-ligated constructs from all nine samples were reported as indicated in the column peak size [bp] . The initial peak at 35 bp and the final peak at 10.380 bp are marker peaks that are system inherent included in all runs.

Techniques Used: Selection, Construct, Marker

8) Product Images from "Investigation of appropriate pre-analytical procedure for circulating free DNA from liquid biopsy"

Article Title: Investigation of appropriate pre-analytical procedure for circulating free DNA from liquid biopsy

Journal: Oncotarget

doi: 10.18632/oncotarget.25881

Influence of anticoagulant and blood preservation conditions on quality of cfDNA from healthy volunteers cfDNA concentrations were examined at the indicated time after blood collection using sodium citrate tubes ( A ) or EDTA 2K tubes ( B ) from ten healthy volunteers. Blood storage temperature until plasma separation was 4° C (white box) or room temperature (gray box). Size distribution of plasma DNA was analyzed with an Agilent bioanalyzer ® ; representative examples are shown in panels C-F. Sodium citrate tubes ( C , E ) or EDTA 2K tubes ( D , F ) were used for blood collection, and blood storage until plasma separation was at RT (C, D) or 4° C (E, F). DNA concentration of 1000 bp to 9000 bp fragments ( G ) and of 100 bp to 250 bp fragments ( H ) in all samples stored at 4° C was measured with an Agilent bioanalyzer ® as described in “Materials and methods”. Blood was collected into sodium citrate tubes (white box) or EDTA 2K tubes (gray box). Statistical analyses were performed with Friedman’s rank test.
Figure Legend Snippet: Influence of anticoagulant and blood preservation conditions on quality of cfDNA from healthy volunteers cfDNA concentrations were examined at the indicated time after blood collection using sodium citrate tubes ( A ) or EDTA 2K tubes ( B ) from ten healthy volunteers. Blood storage temperature until plasma separation was 4° C (white box) or room temperature (gray box). Size distribution of plasma DNA was analyzed with an Agilent bioanalyzer ® ; representative examples are shown in panels C-F. Sodium citrate tubes ( C , E ) or EDTA 2K tubes ( D , F ) were used for blood collection, and blood storage until plasma separation was at RT (C, D) or 4° C (E, F). DNA concentration of 1000 bp to 9000 bp fragments ( G ) and of 100 bp to 250 bp fragments ( H ) in all samples stored at 4° C was measured with an Agilent bioanalyzer ® as described in “Materials and methods”. Blood was collected into sodium citrate tubes (white box) or EDTA 2K tubes (gray box). Statistical analyses were performed with Friedman’s rank test.

Techniques Used: Preserving, Concentration Assay

9) Product Images from "Investigation of appropriate pre-analytical procedure for circulating free DNA from liquid biopsy"

Article Title: Investigation of appropriate pre-analytical procedure for circulating free DNA from liquid biopsy

Journal: Oncotarget

doi: 10.18632/oncotarget.25881

Influence of anticoagulant and blood preservation conditions on quality of cfDNA from healthy volunteers cfDNA concentrations were examined at the indicated time after blood collection using sodium citrate tubes ( A ) or EDTA 2K tubes ( B ) from ten healthy volunteers. Blood storage temperature until plasma separation was 4° C (white box) or room temperature (gray box). Size distribution of plasma DNA was analyzed with an Agilent bioanalyzer ® ; representative examples are shown in panels C-F. Sodium citrate tubes ( C , E ) or EDTA 2K tubes ( D , F ) were used for blood collection, and blood storage until plasma separation was at RT (C, D) or 4° C (E, F). DNA concentration of 1000 bp to 9000 bp fragments ( G ) and of 100 bp to 250 bp fragments ( H ) in all samples stored at 4° C was measured with an Agilent bioanalyzer ® as described in “Materials and methods”. Blood was collected into sodium citrate tubes (white box) or EDTA 2K tubes (gray box). Statistical analyses were performed with Friedman’s rank test.
Figure Legend Snippet: Influence of anticoagulant and blood preservation conditions on quality of cfDNA from healthy volunteers cfDNA concentrations were examined at the indicated time after blood collection using sodium citrate tubes ( A ) or EDTA 2K tubes ( B ) from ten healthy volunteers. Blood storage temperature until plasma separation was 4° C (white box) or room temperature (gray box). Size distribution of plasma DNA was analyzed with an Agilent bioanalyzer ® ; representative examples are shown in panels C-F. Sodium citrate tubes ( C , E ) or EDTA 2K tubes ( D , F ) were used for blood collection, and blood storage until plasma separation was at RT (C, D) or 4° C (E, F). DNA concentration of 1000 bp to 9000 bp fragments ( G ) and of 100 bp to 250 bp fragments ( H ) in all samples stored at 4° C was measured with an Agilent bioanalyzer ® as described in “Materials and methods”. Blood was collected into sodium citrate tubes (white box) or EDTA 2K tubes (gray box). Statistical analyses were performed with Friedman’s rank test.

Techniques Used: Preserving, Concentration Assay

10) Product Images from "Quantification of circulating cell-free DNA to predict patient survival in non-small-cell lung cancer"

Article Title: Quantification of circulating cell-free DNA to predict patient survival in non-small-cell lung cancer

Journal: Oncotarget

doi: 10.18632/oncotarget.21769

Changes in the cfDNA concentration during systemic therapy (A) Change in the cfDNA concentration from baseline to first response assessment, according to the radiological response category. (B) Waterfall plot for the percentage change in the cfDNA concentration at first response assessment. (C) Change in the cfDNA concentration from baseline to the radiological best response, according to the radiological best response category. (D) Waterfall plot for the percentage change in the cfDNA concentration at assessment of radiological best response. cfDNA, cell-free DNA; NSCLC, non-small-cell lung cancer; PR, partial response; SD, stable disease; PD, progression of disease * Kruskal-Wallis test among PR, SD and PD groups ** Data are expressed as the median, followed by the interquartile range in parentheses *** Wilcoxon signed rank test between the cfDNA concentration at baseline and at first follow-up assessment or best response.
Figure Legend Snippet: Changes in the cfDNA concentration during systemic therapy (A) Change in the cfDNA concentration from baseline to first response assessment, according to the radiological response category. (B) Waterfall plot for the percentage change in the cfDNA concentration at first response assessment. (C) Change in the cfDNA concentration from baseline to the radiological best response, according to the radiological best response category. (D) Waterfall plot for the percentage change in the cfDNA concentration at assessment of radiological best response. cfDNA, cell-free DNA; NSCLC, non-small-cell lung cancer; PR, partial response; SD, stable disease; PD, progression of disease * Kruskal-Wallis test among PR, SD and PD groups ** Data are expressed as the median, followed by the interquartile range in parentheses *** Wilcoxon signed rank test between the cfDNA concentration at baseline and at first follow-up assessment or best response.

Techniques Used: Concentration Assay

Study flow diagram cfDNA, cell-free DNA; NSCLC, non-small-cell lung cancer, ADC, adenocarcinoma.
Figure Legend Snippet: Study flow diagram cfDNA, cell-free DNA; NSCLC, non-small-cell lung cancer, ADC, adenocarcinoma.

Techniques Used: Flow Cytometry

Kaplan-Meier estimates of PFS and OS according to the cfDNA concentration in patients with NSCLC (A) PFS and (B) OS according to the baseline cfDNA concentration (≤ 70 ng/mL vs. > 70 ng/mL) in all patients with NSCLC. (C) PFS and (D) OS according to the baseline cfDNA concentration (≤ 70 ng/mL vs. > 70 ng/mL) in chemo-naive patients with stage IV adenocarcinoma. cfDNA, cell-free DNA; NSCLC, non-small-cell lung cancer; PFS, progression-free survival; OS, overall survival.
Figure Legend Snippet: Kaplan-Meier estimates of PFS and OS according to the cfDNA concentration in patients with NSCLC (A) PFS and (B) OS according to the baseline cfDNA concentration (≤ 70 ng/mL vs. > 70 ng/mL) in all patients with NSCLC. (C) PFS and (D) OS according to the baseline cfDNA concentration (≤ 70 ng/mL vs. > 70 ng/mL) in chemo-naive patients with stage IV adenocarcinoma. cfDNA, cell-free DNA; NSCLC, non-small-cell lung cancer; PFS, progression-free survival; OS, overall survival.

Techniques Used: Concentration Assay

Circulating cfDNA time points coded by NSCLC patient identification number Graphical presentation of the association between the cfDNA level and the assessment of radiological response in patients with disease progression (A) and without progression (B) Change in the cfDNA concentration from baseline to best response, according to the radiological best response category; x-axis displays the time to clinical tumor progression. cfDNA, cell-free DNA; NSCLC, non-small-cell lung cancer; PR, partial response; SD, stable disease; PD, progression of disease * Kruskal-Wallis test among PR, SD and PD groups ** Data are expressed as the median, followed by the interquartile range in parentheses *** Wilcoxon signed rank test between the cfDNA concentration at baseline and at best response.
Figure Legend Snippet: Circulating cfDNA time points coded by NSCLC patient identification number Graphical presentation of the association between the cfDNA level and the assessment of radiological response in patients with disease progression (A) and without progression (B) Change in the cfDNA concentration from baseline to best response, according to the radiological best response category; x-axis displays the time to clinical tumor progression. cfDNA, cell-free DNA; NSCLC, non-small-cell lung cancer; PR, partial response; SD, stable disease; PD, progression of disease * Kruskal-Wallis test among PR, SD and PD groups ** Data are expressed as the median, followed by the interquartile range in parentheses *** Wilcoxon signed rank test between the cfDNA concentration at baseline and at best response.

Techniques Used: Concentration Assay

Circulating cfDNA kinetics in patients with NSCLCQuantitative cfDNA dynamics during treatment for NSCLC (A) Change in the cfDNA concentration from baseline to disease progression, according to the radiological best response category. (B) Change in the cfDNA concentration from the previous level to disease progression, according to the radiological best response category. Colors and symbols in the panel represent individual patient cfDNA kinetics; x-axis displays the time to clinical tumor progression. cfDNA, cell-free DNA; NSCLC, non-small-cell lung cancer; PR, partial response; SD, stable disease; PD, progression of disease * Kruskal-Wallis test among PR, SD and PD groups ** Data are expressed as the median, followed by the interquartile range in parentheses. *** Wilcoxon signed rank test between the cfDNA level at disease progression and the baseline or previous cfDNA level.
Figure Legend Snippet: Circulating cfDNA kinetics in patients with NSCLCQuantitative cfDNA dynamics during treatment for NSCLC (A) Change in the cfDNA concentration from baseline to disease progression, according to the radiological best response category. (B) Change in the cfDNA concentration from the previous level to disease progression, according to the radiological best response category. Colors and symbols in the panel represent individual patient cfDNA kinetics; x-axis displays the time to clinical tumor progression. cfDNA, cell-free DNA; NSCLC, non-small-cell lung cancer; PR, partial response; SD, stable disease; PD, progression of disease * Kruskal-Wallis test among PR, SD and PD groups ** Data are expressed as the median, followed by the interquartile range in parentheses. *** Wilcoxon signed rank test between the cfDNA level at disease progression and the baseline or previous cfDNA level.

Techniques Used: Concentration Assay

11) Product Images from "Exosomes maintain cellular homeostasis by excreting harmful DNA from cells"

Article Title: Exosomes maintain cellular homeostasis by excreting harmful DNA from cells

Journal: Nature Communications

doi: 10.1038/ncomms15287

Exosomes contain chromosome fragments. ( a ) transmission electron microscopy micrograph of MVE in pre-senescent TIG-3 cells following immuno-gold labelling for dsDNA. Gold particles are depicted as black dots. Right image shows a digitally zoomed area of exosome. ( b , c ) Exosomal DNA isolated from pre-senescent TIG-3 cells were subjected to size distribution analysis using Electrophoresis Bioanalyzer system ( b ) or to deep sequencing analysis ( c ). Genomic DNA of TIG-3 cells are also subjected to deep sequencing analysis, as control ( c ). The read count of each 500-kb bin was normalized to RPKM and corrected by the mappability ( c ). ( d ) Purified exosomes from pre-senescent TIG-3 cells were subjected to sucrose density-gradient separation followed by western blotting using antibodies shown right, NanoSight analysis (NTA) for quantitative measurement of isolated exosome particles and quantitative PCR analysis for detection of genomic DNA fragments (GRM7 and GPC6).
Figure Legend Snippet: Exosomes contain chromosome fragments. ( a ) transmission electron microscopy micrograph of MVE in pre-senescent TIG-3 cells following immuno-gold labelling for dsDNA. Gold particles are depicted as black dots. Right image shows a digitally zoomed area of exosome. ( b , c ) Exosomal DNA isolated from pre-senescent TIG-3 cells were subjected to size distribution analysis using Electrophoresis Bioanalyzer system ( b ) or to deep sequencing analysis ( c ). Genomic DNA of TIG-3 cells are also subjected to deep sequencing analysis, as control ( c ). The read count of each 500-kb bin was normalized to RPKM and corrected by the mappability ( c ). ( d ) Purified exosomes from pre-senescent TIG-3 cells were subjected to sucrose density-gradient separation followed by western blotting using antibodies shown right, NanoSight analysis (NTA) for quantitative measurement of isolated exosome particles and quantitative PCR analysis for detection of genomic DNA fragments (GRM7 and GPC6).

Techniques Used: Transmission Assay, Electron Microscopy, Isolation, Electrophoresis, Sequencing, Purification, Western Blot, Real-time Polymerase Chain Reaction

12) Product Images from "Exosomes maintain cellular homeostasis by excreting harmful DNA from cells"

Article Title: Exosomes maintain cellular homeostasis by excreting harmful DNA from cells

Journal: Nature Communications

doi: 10.1038/ncomms15287

Exosomes contain chromosome fragments. ( a ) transmission electron microscopy micrograph of MVE in pre-senescent TIG-3 cells following immuno-gold labelling for dsDNA. Gold particles are depicted as black dots. Right image shows a digitally zoomed area of exosome. ( b , c ) Exosomal DNA isolated from pre-senescent TIG-3 cells were subjected to size distribution analysis using Electrophoresis Bioanalyzer system ( b ) or to deep sequencing analysis ( c ). Genomic DNA of TIG-3 cells are also subjected to deep sequencing analysis, as control ( c ). The read count of each 500-kb bin was normalized to RPKM and corrected by the mappability ( c ). ( d ) Purified exosomes from pre-senescent TIG-3 cells were subjected to sucrose density-gradient separation followed by western blotting using antibodies shown right, NanoSight analysis (NTA) for quantitative measurement of isolated exosome particles and quantitative PCR analysis for detection of genomic DNA fragments (GRM7 and GPC6).
Figure Legend Snippet: Exosomes contain chromosome fragments. ( a ) transmission electron microscopy micrograph of MVE in pre-senescent TIG-3 cells following immuno-gold labelling for dsDNA. Gold particles are depicted as black dots. Right image shows a digitally zoomed area of exosome. ( b , c ) Exosomal DNA isolated from pre-senescent TIG-3 cells were subjected to size distribution analysis using Electrophoresis Bioanalyzer system ( b ) or to deep sequencing analysis ( c ). Genomic DNA of TIG-3 cells are also subjected to deep sequencing analysis, as control ( c ). The read count of each 500-kb bin was normalized to RPKM and corrected by the mappability ( c ). ( d ) Purified exosomes from pre-senescent TIG-3 cells were subjected to sucrose density-gradient separation followed by western blotting using antibodies shown right, NanoSight analysis (NTA) for quantitative measurement of isolated exosome particles and quantitative PCR analysis for detection of genomic DNA fragments (GRM7 and GPC6).

Techniques Used: Transmission Assay, Electron Microscopy, Isolation, Electrophoresis, Sequencing, Purification, Western Blot, Real-time Polymerase Chain Reaction

13) Product Images from "Ecophylogeny of the endospheric root fungal microbiome of co-occurring Agrostis stolonifera"

Article Title: Ecophylogeny of the endospheric root fungal microbiome of co-occurring Agrostis stolonifera

Journal: PeerJ

doi: 10.7717/peerj.3454

Phylogenetic tree of the Basidiomycota related root fungal microbiome OTUs. ML tree based on 432 bp of SSU rRNA gene sequences amplified from roots of Agrostis stolonifera . The tree was constructed using representative sequences of the OTUs (taxa without names) and the closest reference sequences (taxa names in italic) from the non-redundant SILVA SSURef ARB database (release 115). Barplots represent the mean expression ratio for each OTU among all samples. Null values indicate that this OTU was not detected in the RNA fraction, value = 1 indicates that this OTU was not detected in the DNA fraction, value = 0.5 indicates that the sum of the relative abundance between DNA and RNA fractions was equal. Green bars: values below 0.5, red bars: values ≥ 0.5. Error bars indicate ±SE. Grey circles indicate the relative abundance of each OTU in the whole dataset. Node support values above 50 are given in the following order: bootstrap values and Bayesian posterior probabilities.
Figure Legend Snippet: Phylogenetic tree of the Basidiomycota related root fungal microbiome OTUs. ML tree based on 432 bp of SSU rRNA gene sequences amplified from roots of Agrostis stolonifera . The tree was constructed using representative sequences of the OTUs (taxa without names) and the closest reference sequences (taxa names in italic) from the non-redundant SILVA SSURef ARB database (release 115). Barplots represent the mean expression ratio for each OTU among all samples. Null values indicate that this OTU was not detected in the RNA fraction, value = 1 indicates that this OTU was not detected in the DNA fraction, value = 0.5 indicates that the sum of the relative abundance between DNA and RNA fractions was equal. Green bars: values below 0.5, red bars: values ≥ 0.5. Error bars indicate ±SE. Grey circles indicate the relative abundance of each OTU in the whole dataset. Node support values above 50 are given in the following order: bootstrap values and Bayesian posterior probabilities.

Techniques Used: Amplification, Construct, Expressing

Phylogenetic tree of the core microbiome OTUs at the level of 19 co-occurring Agrostis stolonifera plants. Tree construction was based on maximum likelihood. Only bootstrap values above 50 are indicated. The tree was constructed using sequences representative of the OTUs (taxa without names) and the closest reference sequences (taxa names in italic) from the non-redundant SILVA SSURef ARB database (release 115). ‘*’: taxa belonging to the ‘DNA core’, ‘ †’: taxa belonging to the ‘RNA core’. Stacked bars indicate the mean relative abundance of each taxon in the DNA (blue) and RNA (red) fractions of the 19 samples. Some taxa belonging to the ‘DNA core’ are also found in the RNA fractions but not in all samples and reciprocally.
Figure Legend Snippet: Phylogenetic tree of the core microbiome OTUs at the level of 19 co-occurring Agrostis stolonifera plants. Tree construction was based on maximum likelihood. Only bootstrap values above 50 are indicated. The tree was constructed using sequences representative of the OTUs (taxa without names) and the closest reference sequences (taxa names in italic) from the non-redundant SILVA SSURef ARB database (release 115). ‘*’: taxa belonging to the ‘DNA core’, ‘ †’: taxa belonging to the ‘RNA core’. Stacked bars indicate the mean relative abundance of each taxon in the DNA (blue) and RNA (red) fractions of the 19 samples. Some taxa belonging to the ‘DNA core’ are also found in the RNA fractions but not in all samples and reciprocally.

Techniques Used: Construct

Alpha diversity and taxonomic composition of fungal communities in DNA and RNA fractions. (A) Distribution of Hill diversity numbers in DNA and RNA sequence data analyses. Different commonly used diversity indexes are special cases of Hill numbers (e.g., q = 0 corresponds to the species richness S, q = 1 corresponds to the exponential of the Shannon-Wiener diversity index, q = 2 corresponds to the inverse Simpson index). Asterisks indicate significantly different means between DNA and RNA fractions (paired t -tests). ‘ns’: p > 0.05, ‘*’: p
Figure Legend Snippet: Alpha diversity and taxonomic composition of fungal communities in DNA and RNA fractions. (A) Distribution of Hill diversity numbers in DNA and RNA sequence data analyses. Different commonly used diversity indexes are special cases of Hill numbers (e.g., q = 0 corresponds to the species richness S, q = 1 corresponds to the exponential of the Shannon-Wiener diversity index, q = 2 corresponds to the inverse Simpson index). Asterisks indicate significantly different means between DNA and RNA fractions (paired t -tests). ‘ns’: p > 0.05, ‘*’: p

Techniques Used: Sequencing

14) Product Images from "Unravelling Intratumoral Heterogeneity through High-Sensitivity Single-Cell Mutational Analysis and Parallel RNA Sequencing"

Article Title: Unravelling Intratumoral Heterogeneity through High-Sensitivity Single-Cell Mutational Analysis and Parallel RNA Sequencing

Journal: Molecular Cell

doi: 10.1016/j.molcel.2019.01.009

TARGET-Seq: A Method for High-Sensitivity Mutation Detection and Parallel Whole-Transcriptome Analysis from the Same Single Cell (A) Schematic representation of the method (full details are available in STAR Methods and Supplemental Experimental Procedures ). In brief, cells were sorted into plates containing TARGET-seq lysis buffer; after lysis, protease was heat inactivated. RT mix was then added. OligodT-ISPCR primed polyadenylated mRNA and target-specific primers primed mRNA molecules of interest. During subsequent PCR, we used ISPCR adaptors to amplify polyA-cDNA, and we used target-specific cDNA and gDNA primers to amplify amplicons of interest. An aliquot of the resulting cDNA+amplicon mix was used for preparing the genotyping library and another aliquot for preparing the transcriptome library for scRNA-seq. (B) Frequency with which TARGET-seq detected heterozygous mutations in ten coding and non-coding regions in cell lines; this approach is compared to SMART-seq+ and mRNA targeting approaches (n = 376 cells, 2–3 independent experiments per amplicon; the bar graph represents mean ± SD). (C) Frequency of detection of heterozygous mutations for the same amplicons as in (B), showing exclusively results from targeted genomic DNA sequencing. The bar graph represents mean ± SD. (D) Frequency of detection of heterozygous mutations in JURKAT cells with SMART-seq+ (n = 36 cells), mRNA targeting (n = 36 cells), gDNA targeting (n = 62 cells), and TARGET-seq (n = 62 cells) when four different mutations ( RUNX1 , NOTCH1 , PTEN , and TP53 ) in the same single cell were profiled in three independent experiments. Each slice of the pie chart represents a different combination of mutations, and each color represents the number of mutations detected per single cell.
Figure Legend Snippet: TARGET-Seq: A Method for High-Sensitivity Mutation Detection and Parallel Whole-Transcriptome Analysis from the Same Single Cell (A) Schematic representation of the method (full details are available in STAR Methods and Supplemental Experimental Procedures ). In brief, cells were sorted into plates containing TARGET-seq lysis buffer; after lysis, protease was heat inactivated. RT mix was then added. OligodT-ISPCR primed polyadenylated mRNA and target-specific primers primed mRNA molecules of interest. During subsequent PCR, we used ISPCR adaptors to amplify polyA-cDNA, and we used target-specific cDNA and gDNA primers to amplify amplicons of interest. An aliquot of the resulting cDNA+amplicon mix was used for preparing the genotyping library and another aliquot for preparing the transcriptome library for scRNA-seq. (B) Frequency with which TARGET-seq detected heterozygous mutations in ten coding and non-coding regions in cell lines; this approach is compared to SMART-seq+ and mRNA targeting approaches (n = 376 cells, 2–3 independent experiments per amplicon; the bar graph represents mean ± SD). (C) Frequency of detection of heterozygous mutations for the same amplicons as in (B), showing exclusively results from targeted genomic DNA sequencing. The bar graph represents mean ± SD. (D) Frequency of detection of heterozygous mutations in JURKAT cells with SMART-seq+ (n = 36 cells), mRNA targeting (n = 36 cells), gDNA targeting (n = 62 cells), and TARGET-seq (n = 62 cells) when four different mutations ( RUNX1 , NOTCH1 , PTEN , and TP53 ) in the same single cell were profiled in three independent experiments. Each slice of the pie chart represents a different combination of mutations, and each color represents the number of mutations detected per single cell.

Techniques Used: Mutagenesis, Lysis, Polymerase Chain Reaction, Amplification, DNA Sequencing

15) Product Images from "Origin of circulating free DNA in patients with lung cancer"

Article Title: Origin of circulating free DNA in patients with lung cancer

Journal: PLoS ONE

doi: 10.1371/journal.pone.0235611

Size distribution of cfDNA analyzed with Agilent Bioanalyzer among patients with lung cancer. (A) Size distribution among 5 selected patients. Size distribution of cfDNA fragments in patients with lung cancer is bimodal with peaks around 170 bp (short) and 5 Kb (long). We defined “short fragment cfDNA” as DNA of size 120~265 bp and “long fragment cfDNA” as 1000~9000 bp. The concentration and molarity of each specified region were normalized by lower (35 bp) and upper (10380 bp) marker buffers. (B) Comparison of molarity of long fragment cfDNA according to presence or absence of distant metastasis. (C) Comparison of molarity of short fragment cfDNA according to presence or absence of distant metastasis.
Figure Legend Snippet: Size distribution of cfDNA analyzed with Agilent Bioanalyzer among patients with lung cancer. (A) Size distribution among 5 selected patients. Size distribution of cfDNA fragments in patients with lung cancer is bimodal with peaks around 170 bp (short) and 5 Kb (long). We defined “short fragment cfDNA” as DNA of size 120~265 bp and “long fragment cfDNA” as 1000~9000 bp. The concentration and molarity of each specified region were normalized by lower (35 bp) and upper (10380 bp) marker buffers. (B) Comparison of molarity of long fragment cfDNA according to presence or absence of distant metastasis. (C) Comparison of molarity of short fragment cfDNA according to presence or absence of distant metastasis.

Techniques Used: Concentration Assay, Marker

16) Product Images from "Multiplex profiling of serum proteins in solution using barcoded antibody fragments and next generation sequencing"

Article Title: Multiplex profiling of serum proteins in solution using barcoded antibody fragments and next generation sequencing

Journal: Communications Biology

doi: 10.1038/s42003-020-1068-0

The concept of the ProMIS assay. a Assay principles (1–4) and the conjugation of scFv with oligonucleotides, using Sortase A (Srt). (1) Biotinylated serum proteins are captured and displayed on streptavidin-coated magnetic beads. (2) Recombinant antibody fragments (scFvs) are site-specifically conjugated 1:1 with a unique DNA oligo containing a scFv-specific tag, using a Sortase A-mediated coupling strategy. The scFv-oligos are then mixed with the beads coated with serum proteins. (3) After washing, adapter PCR is performed to equip bound scFv-oligos with a sample-specific DNA tag. (4) PCR products obtained from the combined scFv and sample tags are finally analyzed, using NGS. b SDS-PAGE analysis of conjugation of scFvs (containing the Sortase A recognition motif LPETG) to tri-glycine modified oligonucleotides, followed by filtration (30 kDa cutoff) to isolate only the conjugated scFv-oligos.
Figure Legend Snippet: The concept of the ProMIS assay. a Assay principles (1–4) and the conjugation of scFv with oligonucleotides, using Sortase A (Srt). (1) Biotinylated serum proteins are captured and displayed on streptavidin-coated magnetic beads. (2) Recombinant antibody fragments (scFvs) are site-specifically conjugated 1:1 with a unique DNA oligo containing a scFv-specific tag, using a Sortase A-mediated coupling strategy. The scFv-oligos are then mixed with the beads coated with serum proteins. (3) After washing, adapter PCR is performed to equip bound scFv-oligos with a sample-specific DNA tag. (4) PCR products obtained from the combined scFv and sample tags are finally analyzed, using NGS. b SDS-PAGE analysis of conjugation of scFvs (containing the Sortase A recognition motif LPETG) to tri-glycine modified oligonucleotides, followed by filtration (30 kDa cutoff) to isolate only the conjugated scFv-oligos.

Techniques Used: Conjugation Assay, Magnetic Beads, Recombinant, Polymerase Chain Reaction, Next-Generation Sequencing, SDS Page, Modification, Filtration

17) Product Images from "Proliferation Cycle Causes Age Dependent Mitochondrial Deficiencies and Contributes to the Aging of Stem Cells"

Article Title: Proliferation Cycle Causes Age Dependent Mitochondrial Deficiencies and Contributes to the Aging of Stem Cells

Journal: Genes

doi: 10.3390/genes8120397

Mitochondrial DNA (mtDNA) rearrangements and impaired mtDNA replication in ovaries of aged flies. ( A ) Schematic drawing of Drosophila melanogaster (Dm.) mtDNA. Enzyme sites of HindIII (H) and XhoI (X) are indicated. Sizes of digested fragments are also labelled. ( B ) Representative gel image of rolling cycle amplification of mtDNA (arrowhead) and DNA marker (M, kb). ( C ) Pattern of rolling cycle amplification (RCA) amplified mtDNA digested by XhoI and HindIII. The 5.8 kb fragment spanning the AT-rich region was recovered for restriction fragment length polymorphism (RFLP) analysis. ( D ) Schematic map of SspI site on 5.8 kb AT-rich region. ( E ) densitometry plot of SspI digestion pattern of 5.8 fragments spanning the AT-rich region from young (2-day-old) and old (60-day-old) ovaries, analyzed by Agilent Bioanalyzer 2100. Note the difference of bands (open arrowheads) demonstrating the rearrangements of AT-rich regions in aged ovaries. DNA dye standard (closed arrowheads) and DNA ladder are marked (M, bp). ( F ) Representative images showing 5-ethynyl-2’-deoxyuridine (EdU) incorporation into mtDNA (green puncta, arrowheads) in ovaries (dashed line, anterior toward left) from young (2-day-old) and old (40-day-old) female flies. Note that the number of EdU puncta was dramatically reduced in germarium from old fly. Arrowhead: mtDNA; arrow: nuclear DNA; scale bar: 10 µm.
Figure Legend Snippet: Mitochondrial DNA (mtDNA) rearrangements and impaired mtDNA replication in ovaries of aged flies. ( A ) Schematic drawing of Drosophila melanogaster (Dm.) mtDNA. Enzyme sites of HindIII (H) and XhoI (X) are indicated. Sizes of digested fragments are also labelled. ( B ) Representative gel image of rolling cycle amplification of mtDNA (arrowhead) and DNA marker (M, kb). ( C ) Pattern of rolling cycle amplification (RCA) amplified mtDNA digested by XhoI and HindIII. The 5.8 kb fragment spanning the AT-rich region was recovered for restriction fragment length polymorphism (RFLP) analysis. ( D ) Schematic map of SspI site on 5.8 kb AT-rich region. ( E ) densitometry plot of SspI digestion pattern of 5.8 fragments spanning the AT-rich region from young (2-day-old) and old (60-day-old) ovaries, analyzed by Agilent Bioanalyzer 2100. Note the difference of bands (open arrowheads) demonstrating the rearrangements of AT-rich regions in aged ovaries. DNA dye standard (closed arrowheads) and DNA ladder are marked (M, bp). ( F ) Representative images showing 5-ethynyl-2’-deoxyuridine (EdU) incorporation into mtDNA (green puncta, arrowheads) in ovaries (dashed line, anterior toward left) from young (2-day-old) and old (40-day-old) female flies. Note that the number of EdU puncta was dramatically reduced in germarium from old fly. Arrowhead: mtDNA; arrow: nuclear DNA; scale bar: 10 µm.

Techniques Used: Amplification, Marker

18) Product Images from "Integrative microRNA and mRNA deep-sequencing expression profiling in endemic Burkitt lymphoma"

Article Title: Integrative microRNA and mRNA deep-sequencing expression profiling in endemic Burkitt lymphoma

Journal: BMC Cancer

doi: 10.1186/s12885-017-3711-9

Aberrant transcriptome expression pivotal to eBL lymphomagenesis. a Schematic illustration of the aberrant gene expression and miRNA mediated regulatory changes that would initiate lymphomagenesis as a result of DNA damage. Combined loss of p53 function due to small interfering RNA-mediated regulation of ATM and NLK together with upregulation of TFAP4, would facilitate survival of cells with the c-myc-Igh chromosomal translocation and MYC induced cell cycle progression initiating eBL tumor development. ATM checkpoint kinase, transduces genomic stress signals to halt cell cycle progression in response to DNA damage. It is critical in the regulation of apoptosis and lymphomagenesis in c-myc induced lymphomas. ATM is downregulated in eBL and it is targeted by 4 miRs that are Upregulated in eBL. NLK is required for the upregulation of P53 expression in response to DNA damage. It interacts with P53 to enhance its stability and activity by abrogating MDM2 mediated degradation. NLK is downregulated in eBL tumor cells and also targeted by 2 miRs that are upregulated in eBL tumor cells. TFAP4/AP4 is a central mediator of cell cycle progression in response to c-MYC activation. b RNA seq. Expression counts of MYC , TFAP4 , ATM and NLK in eBL tumor cells and GC B cells. c Hierarchical clustering of eBL and GC B cells based on the expression profiles of MYC , TFAP4 , ATM and NLK also revealed a clear separation of the two groups. d . miRNA seq. Expression counts of hsa-miR-26a-5p, hsa-miR-27b-3p, hsa-miR-30b-5p, miR-17~92-cluster members (hsa-miR-19b-3p, and hsa-miR-92a-3p), and let-7-family miRs (hsa-let-7a-5p, hsa-let-7b-5p, hsa-let-7d-5p, hsa-let-7e-5p, and hsa-let-7 g-5p) in eBL tumor cells and GC B cells
Figure Legend Snippet: Aberrant transcriptome expression pivotal to eBL lymphomagenesis. a Schematic illustration of the aberrant gene expression and miRNA mediated regulatory changes that would initiate lymphomagenesis as a result of DNA damage. Combined loss of p53 function due to small interfering RNA-mediated regulation of ATM and NLK together with upregulation of TFAP4, would facilitate survival of cells with the c-myc-Igh chromosomal translocation and MYC induced cell cycle progression initiating eBL tumor development. ATM checkpoint kinase, transduces genomic stress signals to halt cell cycle progression in response to DNA damage. It is critical in the regulation of apoptosis and lymphomagenesis in c-myc induced lymphomas. ATM is downregulated in eBL and it is targeted by 4 miRs that are Upregulated in eBL. NLK is required for the upregulation of P53 expression in response to DNA damage. It interacts with P53 to enhance its stability and activity by abrogating MDM2 mediated degradation. NLK is downregulated in eBL tumor cells and also targeted by 2 miRs that are upregulated in eBL tumor cells. TFAP4/AP4 is a central mediator of cell cycle progression in response to c-MYC activation. b RNA seq. Expression counts of MYC , TFAP4 , ATM and NLK in eBL tumor cells and GC B cells. c Hierarchical clustering of eBL and GC B cells based on the expression profiles of MYC , TFAP4 , ATM and NLK also revealed a clear separation of the two groups. d . miRNA seq. Expression counts of hsa-miR-26a-5p, hsa-miR-27b-3p, hsa-miR-30b-5p, miR-17~92-cluster members (hsa-miR-19b-3p, and hsa-miR-92a-3p), and let-7-family miRs (hsa-let-7a-5p, hsa-let-7b-5p, hsa-let-7d-5p, hsa-let-7e-5p, and hsa-let-7 g-5p) in eBL tumor cells and GC B cells

Techniques Used: Expressing, Small Interfering RNA, Translocation Assay, Activity Assay, Activation Assay, RNA Sequencing Assay

19) Product Images from "Methodology for Y Chromosome Capture: A complete genome sequence of  Y chromosome using flow cytometry, laser microdissection and magnetic streptavidin-beads"

Article Title: Methodology for Y Chromosome Capture: A complete genome sequence of  Y chromosome using flow cytometry, laser microdissection and magnetic streptavidin-beads

Journal: Scientific Reports

doi: 10.1038/s41598-018-27819-x

( A ) Microfluidic electrophoretic separation of the different methods of Y chromosome capture using a DNA high sensitivity (HS) Bioanalyzer assay. Sample 1: flow cytometry capture, Sample 2: laser capture microdissection, Sample 3: magnetic streptavidin-bead capture. The HS ladder (on left) ranges from 35 base pair (bp) to 7000 bp. All sample peaks appear between the lower and upper marker peaks (35–10380 bp). ( B ) Bioanalyzer high sensitivity profiles of each capture technique. The protocol for this assay is as follows: The captured DNA (putative Y chromosome) was sonicated to a size between 150 and 500 bp and after the sonication DNA was loaded on the Bioanalyzer assay. The sonication program (see 4.5.1. DNA Shearing) was tested previously to obtain the specific fragment size, which has been verified to be proper for preparing a DNA library.
Figure Legend Snippet: ( A ) Microfluidic electrophoretic separation of the different methods of Y chromosome capture using a DNA high sensitivity (HS) Bioanalyzer assay. Sample 1: flow cytometry capture, Sample 2: laser capture microdissection, Sample 3: magnetic streptavidin-bead capture. The HS ladder (on left) ranges from 35 base pair (bp) to 7000 bp. All sample peaks appear between the lower and upper marker peaks (35–10380 bp). ( B ) Bioanalyzer high sensitivity profiles of each capture technique. The protocol for this assay is as follows: The captured DNA (putative Y chromosome) was sonicated to a size between 150 and 500 bp and after the sonication DNA was loaded on the Bioanalyzer assay. The sonication program (see 4.5.1. DNA Shearing) was tested previously to obtain the specific fragment size, which has been verified to be proper for preparing a DNA library.

Techniques Used: Flow Cytometry, Cytometry, Laser Capture Microdissection, Marker, Sonication

20) Product Images from "Generating Exome Enriched Sequencing Libraries from Formalin-Fixed, Paraffin-Embedded Tissue DNA for Next Generation Sequencing"

Article Title: Generating Exome Enriched Sequencing Libraries from Formalin-Fixed, Paraffin-Embedded Tissue DNA for Next Generation Sequencing

Journal: Current protocols in human genetics

doi: 10.1002/cphg.27

DNA fragmentation quality check. 50ng of FFPE derived DNA was sheared using the Covaris E220 instrument. Following clean up each sample was diluted 1:5 and run on a 2100 BioAnalyzer High Sensitivity DNA chip to check the DNA fragment size distribution and sample concentration. A broad distribution of sizes should be expected between 100-2000bp is typical, with the majority of the fragments in the 200-800bp range.
Figure Legend Snippet: DNA fragmentation quality check. 50ng of FFPE derived DNA was sheared using the Covaris E220 instrument. Following clean up each sample was diluted 1:5 and run on a 2100 BioAnalyzer High Sensitivity DNA chip to check the DNA fragment size distribution and sample concentration. A broad distribution of sizes should be expected between 100-2000bp is typical, with the majority of the fragments in the 200-800bp range.

Techniques Used: Formalin-fixed Paraffin-Embedded, Derivative Assay, Chromatin Immunoprecipitation, Concentration Assay

Pre-Capture amplification quality check. Adapter ligated libraries were amplified prior to hybridization capture. Following clean up each sample was diluted 1:100 and run on a 2100 BioAnalyzer High Sensitivity DNA chip to confirm library fragment size and concentration. A focused size distribution between 200-800bp is typical. Quality of FFPE samples may affect how this distribution appears (average size between 250-550bp). Fragment size includes the added length of bases (123bp) from the ligated adapters.
Figure Legend Snippet: Pre-Capture amplification quality check. Adapter ligated libraries were amplified prior to hybridization capture. Following clean up each sample was diluted 1:100 and run on a 2100 BioAnalyzer High Sensitivity DNA chip to confirm library fragment size and concentration. A focused size distribution between 200-800bp is typical. Quality of FFPE samples may affect how this distribution appears (average size between 250-550bp). Fragment size includes the added length of bases (123bp) from the ligated adapters.

Techniques Used: Amplification, Hybridization, Chromatin Immunoprecipitation, Concentration Assay, Formalin-fixed Paraffin-Embedded

21) Product Images from "Generating Exome Enriched Sequencing Libraries from Formalin-Fixed, Paraffin-Embedded Tissue DNA for Next Generation Sequencing"

Article Title: Generating Exome Enriched Sequencing Libraries from Formalin-Fixed, Paraffin-Embedded Tissue DNA for Next Generation Sequencing

Journal: Current protocols in human genetics

doi: 10.1002/cphg.27

DNA fragmentation quality check. 50ng of FFPE derived DNA was sheared using the Covaris E220 instrument. Following clean up each sample was diluted 1:5 and run on a 2100 BioAnalyzer High Sensitivity DNA chip to check the DNA fragment size distribution and sample concentration. A broad distribution of sizes should be expected between 100-2000bp is typical, with the majority of the fragments in the 200-800bp range.
Figure Legend Snippet: DNA fragmentation quality check. 50ng of FFPE derived DNA was sheared using the Covaris E220 instrument. Following clean up each sample was diluted 1:5 and run on a 2100 BioAnalyzer High Sensitivity DNA chip to check the DNA fragment size distribution and sample concentration. A broad distribution of sizes should be expected between 100-2000bp is typical, with the majority of the fragments in the 200-800bp range.

Techniques Used: Formalin-fixed Paraffin-Embedded, Derivative Assay, Chromatin Immunoprecipitation, Concentration Assay

Pre-Capture amplification quality check. Adapter ligated libraries were amplified prior to hybridization capture. Following clean up each sample was diluted 1:100 and run on a 2100 BioAnalyzer High Sensitivity DNA chip to confirm library fragment size and concentration. A focused size distribution between 200-800bp is typical. Quality of FFPE samples may affect how this distribution appears (average size between 250-550bp). Fragment size includes the added length of bases (123bp) from the ligated adapters.
Figure Legend Snippet: Pre-Capture amplification quality check. Adapter ligated libraries were amplified prior to hybridization capture. Following clean up each sample was diluted 1:100 and run on a 2100 BioAnalyzer High Sensitivity DNA chip to confirm library fragment size and concentration. A focused size distribution between 200-800bp is typical. Quality of FFPE samples may affect how this distribution appears (average size between 250-550bp). Fragment size includes the added length of bases (123bp) from the ligated adapters.

Techniques Used: Amplification, Hybridization, Chromatin Immunoprecipitation, Concentration Assay, Formalin-fixed Paraffin-Embedded

22) Product Images from "Improved Protocols for Illumina Sequencing"

Article Title: Improved Protocols for Illumina Sequencing

Journal: Current protocols in human genetics / editorial board, Jonathan L. Haines ... [et al.]

doi: 10.1002/0471142905.hg1802s62

Effect of AMPure XP ratios on fragment size selection. 1 μg of DNA was sheared to giver fragments from 20 to 400bp (average 160bp). Next the DNA was incubated with different AMPure ratios. The size distribution of fragments after AMPure clean up were analysed by electrophoresis using an Agilent Bioanalyzer High Sensitivity DNA chip. (A) AMPure bead to DNA ratios varying from 2.5× beads to DNA up to 1.0× beads to DNA (B) AMPure bead to DNA ratios varying from 1.5× beads to DNA up to 0.6× beads to DNA.
Figure Legend Snippet: Effect of AMPure XP ratios on fragment size selection. 1 μg of DNA was sheared to giver fragments from 20 to 400bp (average 160bp). Next the DNA was incubated with different AMPure ratios. The size distribution of fragments after AMPure clean up were analysed by electrophoresis using an Agilent Bioanalyzer High Sensitivity DNA chip. (A) AMPure bead to DNA ratios varying from 2.5× beads to DNA up to 1.0× beads to DNA (B) AMPure bead to DNA ratios varying from 1.5× beads to DNA up to 0.6× beads to DNA.

Techniques Used: Selection, Incubation, Electrophoresis, Chromatin Immunoprecipitation

23) Product Images from "Quartz-Seq2: a high-throughput single-cell RNA-sequencing method that effectively uses limited sequence reads"

Article Title: Quartz-Seq2: a high-throughput single-cell RNA-sequencing method that effectively uses limited sequence reads

Journal: Genome Biology

doi: 10.1186/s13059-018-1407-3

Overview of Quartz-Seq2 experimental processes. a Quartz-Seq2 consists of five steps. (1) Each single cell in a droplet is sorted into lysis buffer in each well of a 384-well PCR plate using flow cytometry analysis data. (2) Poly-adenylated RNA in each well is reverse-transcribed into first-strand cDNA with reverse transcription primer, which has a unique cell barcode ( CB ). We prepare 384 or 1536 kinds of cell barcode with a unique sequence based on the Sequence–Levenshtein distance (SeqLv). The edit distance of SeqLv is 5. The RT primer also has a UMI sequence for reduction of PCR bias (MB) and a poly(dT) sequence for binding to poly(A) RNA. (3) Cell barcode-labeled cDNAs from all 384 wells are promptly collected by centrifugation using assembled collectors. (4) Collected first-strand cDNAs are purified and concentrated for subsequent whole-transcript amplification. In the poly(A) tailing step, purified cDNA is extended with a poly(A) tail by terminal deoxynucleotidyl transferase ( TdT ). Subsequently, second-strand cDNA is synthesized with a tagging primer, which has a poly(dT) sequence. The resulting second-strand cDNA has a PCR primer sequence ( M ) at both ends of it. The cDNA is amplifiable in a subsequent PCR amplification. (5) For conversion from amplified cDNA to sequence library DNA, we fragment the amplified cDNA using the ultrasonicator Covaris. Such fragmented cDNA is ligated with a truncated Y-shaped sequence adaptor, which has an Illumina flow-cell binding sequence ( P7 ) and a pool barcode sequence ( PB ). The PB makes it possible to mix different sets of cell barcode-labeled cDNA. Ligated cDNA, which has CB and MB sequences, is enriched by PCR amplification. The resulting sequence library DNA contains P7 and P5 flow-cell binding sequences at respective ends of the DNA. We sequence the cell barcode site and the UMI site at Read1, the pool barcode site at Index1, and the transcript sequence at Read2. b The relationship between initial fastq reads and the number of single cells for sequence analysis in NextSeq500 runs. Typically, one sequence run with NextSeq 500/550 High Output v2 Kit reads out 400–450 M fastq reads. The x-axis represents the input cell number for one sequence run. The y-axis represents the initial data size (fastq reads) on average per cell. The red outline represents the typical range of shallow input read depth for a single cell. c We define the formula for calculating the UMI conversion efficiency. Each parameter is defined as follows: UMI sc is the number of UMI counts, assigned to a single-cell sample, fastq sc is the number of fastq reads derived from each single-cell sample, fastq non-sc is the number of fastq reads derived from non-single-cell samples, which include experimental byproducts such as WTA adaptors, WTA byproducts, and non-STAMPs. Initial fastq reads are composed of fastq sc and fastq non-sc
Figure Legend Snippet: Overview of Quartz-Seq2 experimental processes. a Quartz-Seq2 consists of five steps. (1) Each single cell in a droplet is sorted into lysis buffer in each well of a 384-well PCR plate using flow cytometry analysis data. (2) Poly-adenylated RNA in each well is reverse-transcribed into first-strand cDNA with reverse transcription primer, which has a unique cell barcode ( CB ). We prepare 384 or 1536 kinds of cell barcode with a unique sequence based on the Sequence–Levenshtein distance (SeqLv). The edit distance of SeqLv is 5. The RT primer also has a UMI sequence for reduction of PCR bias (MB) and a poly(dT) sequence for binding to poly(A) RNA. (3) Cell barcode-labeled cDNAs from all 384 wells are promptly collected by centrifugation using assembled collectors. (4) Collected first-strand cDNAs are purified and concentrated for subsequent whole-transcript amplification. In the poly(A) tailing step, purified cDNA is extended with a poly(A) tail by terminal deoxynucleotidyl transferase ( TdT ). Subsequently, second-strand cDNA is synthesized with a tagging primer, which has a poly(dT) sequence. The resulting second-strand cDNA has a PCR primer sequence ( M ) at both ends of it. The cDNA is amplifiable in a subsequent PCR amplification. (5) For conversion from amplified cDNA to sequence library DNA, we fragment the amplified cDNA using the ultrasonicator Covaris. Such fragmented cDNA is ligated with a truncated Y-shaped sequence adaptor, which has an Illumina flow-cell binding sequence ( P7 ) and a pool barcode sequence ( PB ). The PB makes it possible to mix different sets of cell barcode-labeled cDNA. Ligated cDNA, which has CB and MB sequences, is enriched by PCR amplification. The resulting sequence library DNA contains P7 and P5 flow-cell binding sequences at respective ends of the DNA. We sequence the cell barcode site and the UMI site at Read1, the pool barcode site at Index1, and the transcript sequence at Read2. b The relationship between initial fastq reads and the number of single cells for sequence analysis in NextSeq500 runs. Typically, one sequence run with NextSeq 500/550 High Output v2 Kit reads out 400–450 M fastq reads. The x-axis represents the input cell number for one sequence run. The y-axis represents the initial data size (fastq reads) on average per cell. The red outline represents the typical range of shallow input read depth for a single cell. c We define the formula for calculating the UMI conversion efficiency. Each parameter is defined as follows: UMI sc is the number of UMI counts, assigned to a single-cell sample, fastq sc is the number of fastq reads derived from each single-cell sample, fastq non-sc is the number of fastq reads derived from non-single-cell samples, which include experimental byproducts such as WTA adaptors, WTA byproducts, and non-STAMPs. Initial fastq reads are composed of fastq sc and fastq non-sc

Techniques Used: Lysis, Polymerase Chain Reaction, Flow Cytometry, Cytometry, Sequencing, Binding Assay, Labeling, Centrifugation, Purification, Amplification, Synthesized, Derivative Assay

Sequence performance of Quartz-Seq2 with molecular biological improvements. a Improvement of poly(A) tagging efficiency. The relative DNA yield in various poly(A) tagging conditions using purified first-strand cDNA from 1 ng of total RNA. T55 buffer as the terminal deoxynucleotidyl transferase (TdT) buffer and the temperature condition “ Increment : Table S5. QuartzB represents use of a Quartz-Seq-like buffer as a positive control, in accordance with the approach described in the original Quartz-Seq paper. Finally, we quantified cDNA yield (300–9000 bp) and byproduct DNA yield (50–300 bp) using a Bioanalyzer (Agilent). The presented p value was obtained using two-tailed Welch’s t -test. b Reverse transcription efficiency with serially diluted RT enzymes. The x-axis : Figure S7. c , f – h Comparison between Quartz-Seq2 in the RT25 condition and Quartz-Seq-like conditions regarding sequence performance. c We analyzed 384 wells with 10 pg of total RNA and used approximately 0.19 M fastq reads on average per well. We show the UMI count and gene count in box plots. d A scatter plot between the mean of gene expression and the variability of gene expression with 10 pg of total RNA in 384 wells. Red lines represent the theoretical variability of gene expression in the form of a Poisson distribution. e Gene expression reproducibility between bulk poly(A)-RNA-seq (1 μg of total RNA) and Quartz-Seq2 (10 pg of total RNA, averaged over 384 wells). f Dispersion of gene expression. The x-axis represents gene expression variability. g Reproducibility of gene expression for internal gene and external control RNA. h Accuracy of gene expression for internal gene and external control RNA
Figure Legend Snippet: Sequence performance of Quartz-Seq2 with molecular biological improvements. a Improvement of poly(A) tagging efficiency. The relative DNA yield in various poly(A) tagging conditions using purified first-strand cDNA from 1 ng of total RNA. T55 buffer as the terminal deoxynucleotidyl transferase (TdT) buffer and the temperature condition “ Increment : Table S5. QuartzB represents use of a Quartz-Seq-like buffer as a positive control, in accordance with the approach described in the original Quartz-Seq paper. Finally, we quantified cDNA yield (300–9000 bp) and byproduct DNA yield (50–300 bp) using a Bioanalyzer (Agilent). The presented p value was obtained using two-tailed Welch’s t -test. b Reverse transcription efficiency with serially diluted RT enzymes. The x-axis : Figure S7. c , f – h Comparison between Quartz-Seq2 in the RT25 condition and Quartz-Seq-like conditions regarding sequence performance. c We analyzed 384 wells with 10 pg of total RNA and used approximately 0.19 M fastq reads on average per well. We show the UMI count and gene count in box plots. d A scatter plot between the mean of gene expression and the variability of gene expression with 10 pg of total RNA in 384 wells. Red lines represent the theoretical variability of gene expression in the form of a Poisson distribution. e Gene expression reproducibility between bulk poly(A)-RNA-seq (1 μg of total RNA) and Quartz-Seq2 (10 pg of total RNA, averaged over 384 wells). f Dispersion of gene expression. The x-axis represents gene expression variability. g Reproducibility of gene expression for internal gene and external control RNA. h Accuracy of gene expression for internal gene and external control RNA

Techniques Used: Sequencing, Purification, Positive Control, Two Tailed Test, Expressing, RNA Sequencing Assay

24) Product Images from "Unlocking Short Read Sequencing for Metagenomics"

Article Title: Unlocking Short Read Sequencing for Metagenomics

Journal: PLoS ONE

doi: 10.1371/journal.pone.0011840

Reproducibility of double-SPRI. The panel shows DNA fragment size distributions as obtained by Bioanalyzer DNA-1000 assays. The curves represent the size fractions removed during the first separation step A ) or recovered after the second separation B ). The two size fractions were independently reproduced in 4 or 8 separation experiments. The curves in B ) represent the libraries sequenced after dSPRI based size selection, adapter ligation and PCR enrichment. While concentrations (arbitrary fluorescence units) vary between reproduced libraries the range of removed or enriched DNA fragment sizes was highly reproducible. Panel c ) shows the DNA fragment size distribution recovered after the second separation when using decreasing amounts sheared genomic DNA. dSPRI allows reliable size selection in a DNA concentration independent manner.
Figure Legend Snippet: Reproducibility of double-SPRI. The panel shows DNA fragment size distributions as obtained by Bioanalyzer DNA-1000 assays. The curves represent the size fractions removed during the first separation step A ) or recovered after the second separation B ). The two size fractions were independently reproduced in 4 or 8 separation experiments. The curves in B ) represent the libraries sequenced after dSPRI based size selection, adapter ligation and PCR enrichment. While concentrations (arbitrary fluorescence units) vary between reproduced libraries the range of removed or enriched DNA fragment sizes was highly reproducible. Panel c ) shows the DNA fragment size distribution recovered after the second separation when using decreasing amounts sheared genomic DNA. dSPRI allows reliable size selection in a DNA concentration independent manner.

Techniques Used: Selection, Ligation, Polymerase Chain Reaction, Fluorescence, Concentration Assay

Size-dependent isolation of DNA fragments from sheared genomic DNA via dSPRI. AMPure XP SPRI beads bind DNA fragments in a size dependent manner according to the concentration of salts and polyethylene glycol (PEG) in the reaction [4] – [7] , which can easily be changed by using different volume ratios of DNA to SPRI bead solutions. A two-step procedure is employed to isolate targeted DNA size fractions. Panels A to H present Bioanalyzer DNA-1000 assays showing the sheared genomic DNA used as starting material (black), the larger size DNA fragments discarded in separation 1 (red), and the size fraction purified and recovered after separation 2 (blue). Panel I is a table summarizing the conditions and results displayed in panels A to H. All Bioanalyzer DNA-1000 traces after separation 1 (panel J), and after separation 2 (panel K), are respectively displayed on a graph for the conditions presented in panels A to H. The conditions displayed in panel H were used to obtaine the Illumina composite reads discussed in the text. The wider DNA fragment size distribution from panel H allowed to better analyze the effects of shorter versus longer overlapping regions on consensus reads.
Figure Legend Snippet: Size-dependent isolation of DNA fragments from sheared genomic DNA via dSPRI. AMPure XP SPRI beads bind DNA fragments in a size dependent manner according to the concentration of salts and polyethylene glycol (PEG) in the reaction [4] – [7] , which can easily be changed by using different volume ratios of DNA to SPRI bead solutions. A two-step procedure is employed to isolate targeted DNA size fractions. Panels A to H present Bioanalyzer DNA-1000 assays showing the sheared genomic DNA used as starting material (black), the larger size DNA fragments discarded in separation 1 (red), and the size fraction purified and recovered after separation 2 (blue). Panel I is a table summarizing the conditions and results displayed in panels A to H. All Bioanalyzer DNA-1000 traces after separation 1 (panel J), and after separation 2 (panel K), are respectively displayed on a graph for the conditions presented in panels A to H. The conditions displayed in panel H were used to obtaine the Illumina composite reads discussed in the text. The wider DNA fragment size distribution from panel H allowed to better analyze the effects of shorter versus longer overlapping regions on consensus reads.

Techniques Used: Isolation, Concentration Assay, Purification

25) Product Images from "Nucleotide-resolution DNA double-strand breaks mapping by next-generation sequencing"

Article Title: Nucleotide-resolution DNA double-strand breaks mapping by next-generation sequencing

Journal: Nature methods

doi: 10.1038/nmeth.2408

ASRs validation. ( a ) Fraction of input DNA captured by ChIP in regions with significant (grey highlight) vs. non-significant aphidicolin effect in HeLa cells treated (orange) or not (green) with aphidicolin. Mean ± s.d. are shown for n = 3 biological replicates. Genomic coordinates of amplicons analyzed by qPCR are reported. Chr: chromosome. Coord: genomic coordinate. ( b ) Comparison of aphidicolin effect measured by BLESS vs. ChIP in regions described in ( a ). Captured DNA ratio: ratio of captured DNA in aphidicolin-treated (A) vs. control (C) HeLa. R: Pearson’s correlation coefficient.
Figure Legend Snippet: ASRs validation. ( a ) Fraction of input DNA captured by ChIP in regions with significant (grey highlight) vs. non-significant aphidicolin effect in HeLa cells treated (orange) or not (green) with aphidicolin. Mean ± s.d. are shown for n = 3 biological replicates. Genomic coordinates of amplicons analyzed by qPCR are reported. Chr: chromosome. Coord: genomic coordinate. ( b ) Comparison of aphidicolin effect measured by BLESS vs. ChIP in regions described in ( a ). Captured DNA ratio: ratio of captured DNA in aphidicolin-treated (A) vs. control (C) HeLa. R: Pearson’s correlation coefficient.

Techniques Used: Chromatin Immunoprecipitation, Real-time Polymerase Chain Reaction

26) Product Images from "Highly sensitive and full-genome interrogation of SARS-CoV-2 using multiplexed PCR enrichment followed by next-generation sequencing"

Article Title: Highly sensitive and full-genome interrogation of SARS-CoV-2 using multiplexed PCR enrichment followed by next-generation sequencing

Journal: bioRxiv

doi: 10.1101/2020.03.12.988246

Comparison of LOD between multiplex PCR and regular PCR. A total of 35 cycles was used in multiplex PCR, while 45 cycles were used for regular PCR. The resulting amplification products from multiplex PCR were processed as described in the Materials and Methods. The PCR products were directly resolved using high sensitivity DNA chips on a Bioanalyzer 2100 instrument. X-axis indicates fragment size (bp) and y-axis indicates fluorescence units. The arrows point to the expected specific amplification products. The number of plasmid copies is indicated on the left.
Figure Legend Snippet: Comparison of LOD between multiplex PCR and regular PCR. A total of 35 cycles was used in multiplex PCR, while 45 cycles were used for regular PCR. The resulting amplification products from multiplex PCR were processed as described in the Materials and Methods. The PCR products were directly resolved using high sensitivity DNA chips on a Bioanalyzer 2100 instrument. X-axis indicates fragment size (bp) and y-axis indicates fluorescence units. The arrows point to the expected specific amplification products. The number of plasmid copies is indicated on the left.

Techniques Used: Multiplex Assay, Polymerase Chain Reaction, Amplification, Fluorescence, Plasmid Preparation

27) Product Images from "Genome-wide mapping of embedded ribonucleotides and other non-canonical nucleotides using emRiboSeq and EndoSeq"

Article Title: Genome-wide mapping of embedded ribonucleotides and other non-canonical nucleotides using emRiboSeq and EndoSeq

Journal: Nature protocols

doi: 10.1038/nprot.2015.099

Library quality control and anticipated results. ( a ) Sonicated DNA separated by agarose gel electrophoresis (Step 25) shows an average fragment size of approximately 400 bp. ( b ) Bioanalyzer result (Step 84) for an emRiboSeq library shows a typical trace (left) and gel-like image (right) with a peak for fragments between ˜180 and ˜300 bp in size (black bar). Standards (green and purple bars) of defined size and amount allow quantification. FU, arbitrary fluorescence units. ( c ) Agarose gel electrophoresis of PCR products after 15, 16 and 17 cycles of amplification (Steps 81-83) of the same library shows product between 200 and 300 bp in size. ( d ) Sequencing results for libraries generated using Nb.BtsI are highly reproducibility between different strains (POL, wildtype polymerase; pol1-L868M, increased Pol-α ribonucleotide incorporation) after normalizing read counts to sequence tags per million (TPM). The majority of bona fide Nb.BtsI sites were present at maximal frequency, although some sites were present at lower frequencies. This is the result of partial loss during size selection because of their close proximity to other cleavage sites, a highly reproducible finding between independent libraries (Spearman's rho=0.82, p
Figure Legend Snippet: Library quality control and anticipated results. ( a ) Sonicated DNA separated by agarose gel electrophoresis (Step 25) shows an average fragment size of approximately 400 bp. ( b ) Bioanalyzer result (Step 84) for an emRiboSeq library shows a typical trace (left) and gel-like image (right) with a peak for fragments between ˜180 and ˜300 bp in size (black bar). Standards (green and purple bars) of defined size and amount allow quantification. FU, arbitrary fluorescence units. ( c ) Agarose gel electrophoresis of PCR products after 15, 16 and 17 cycles of amplification (Steps 81-83) of the same library shows product between 200 and 300 bp in size. ( d ) Sequencing results for libraries generated using Nb.BtsI are highly reproducibility between different strains (POL, wildtype polymerase; pol1-L868M, increased Pol-α ribonucleotide incorporation) after normalizing read counts to sequence tags per million (TPM). The majority of bona fide Nb.BtsI sites were present at maximal frequency, although some sites were present at lower frequencies. This is the result of partial loss during size selection because of their close proximity to other cleavage sites, a highly reproducible finding between independent libraries (Spearman's rho=0.82, p

Techniques Used: Sonication, Agarose Gel Electrophoresis, Fluorescence, Polymerase Chain Reaction, Amplification, Sequencing, Generated, Selection

28) Product Images from "Lack of association between the pancreatitis risk allele CEL-HYB and pancreatic cancer"

Article Title: Lack of association between the pancreatitis risk allele CEL-HYB and pancreatic cancer

Journal: Oncotarget

doi: 10.18632/oncotarget.15137

Bioanalyzer results of pooled 1 st PCR products A. HPDE showed only one band whereas the positive control, PTC1, had one extra band. Sample 6 (circle) shows the detection of an extra band when the positive control DNA was mixed with 3 negative control DNA samples. B. Examples of CEL-HYB positive and negative samples identified by Bioanalyzer. A pool mixed with one positive case and three negative cases showed two bands (Sample 2). Individual analysis of each of these four samples detected the positive case (Sample 4), and three negative cases (Sample 3, 5, and 6).
Figure Legend Snippet: Bioanalyzer results of pooled 1 st PCR products A. HPDE showed only one band whereas the positive control, PTC1, had one extra band. Sample 6 (circle) shows the detection of an extra band when the positive control DNA was mixed with 3 negative control DNA samples. B. Examples of CEL-HYB positive and negative samples identified by Bioanalyzer. A pool mixed with one positive case and three negative cases showed two bands (Sample 2). Individual analysis of each of these four samples detected the positive case (Sample 4), and three negative cases (Sample 3, 5, and 6).

Techniques Used: Polymerase Chain Reaction, Positive Control, Negative Control

29) Product Images from "Quantification of circulating cell-free DNA to predict patient survival in non-small-cell lung cancer"

Article Title: Quantification of circulating cell-free DNA to predict patient survival in non-small-cell lung cancer

Journal: Oncotarget

doi: 10.18632/oncotarget.21769

Changes in the cfDNA concentration during systemic therapy (A) Change in the cfDNA concentration from baseline to first response assessment, according to the radiological response category. (B) Waterfall plot for the percentage change in the cfDNA concentration at first response assessment. (C) Change in the cfDNA concentration from baseline to the radiological best response, according to the radiological best response category. (D) Waterfall plot for the percentage change in the cfDNA concentration at assessment of radiological best response. cfDNA, cell-free DNA; NSCLC, non-small-cell lung cancer; PR, partial response; SD, stable disease; PD, progression of disease * Kruskal-Wallis test among PR, SD and PD groups ** Data are expressed as the median, followed by the interquartile range in parentheses *** Wilcoxon signed rank test between the cfDNA concentration at baseline and at first follow-up assessment or best response.
Figure Legend Snippet: Changes in the cfDNA concentration during systemic therapy (A) Change in the cfDNA concentration from baseline to first response assessment, according to the radiological response category. (B) Waterfall plot for the percentage change in the cfDNA concentration at first response assessment. (C) Change in the cfDNA concentration from baseline to the radiological best response, according to the radiological best response category. (D) Waterfall plot for the percentage change in the cfDNA concentration at assessment of radiological best response. cfDNA, cell-free DNA; NSCLC, non-small-cell lung cancer; PR, partial response; SD, stable disease; PD, progression of disease * Kruskal-Wallis test among PR, SD and PD groups ** Data are expressed as the median, followed by the interquartile range in parentheses *** Wilcoxon signed rank test between the cfDNA concentration at baseline and at first follow-up assessment or best response.

Techniques Used: Concentration Assay

Study flow diagram cfDNA, cell-free DNA; NSCLC, non-small-cell lung cancer, ADC, adenocarcinoma.
Figure Legend Snippet: Study flow diagram cfDNA, cell-free DNA; NSCLC, non-small-cell lung cancer, ADC, adenocarcinoma.

Techniques Used: Flow Cytometry

Kaplan-Meier estimates of PFS and OS according to the cfDNA concentration in patients with NSCLC (A) PFS and (B) OS according to the baseline cfDNA concentration (≤ 70 ng/mL vs. > 70 ng/mL) in all patients with NSCLC. (C) PFS and (D) OS according to the baseline cfDNA concentration (≤ 70 ng/mL vs. > 70 ng/mL) in chemo-naive patients with stage IV adenocarcinoma. cfDNA, cell-free DNA; NSCLC, non-small-cell lung cancer; PFS, progression-free survival; OS, overall survival.
Figure Legend Snippet: Kaplan-Meier estimates of PFS and OS according to the cfDNA concentration in patients with NSCLC (A) PFS and (B) OS according to the baseline cfDNA concentration (≤ 70 ng/mL vs. > 70 ng/mL) in all patients with NSCLC. (C) PFS and (D) OS according to the baseline cfDNA concentration (≤ 70 ng/mL vs. > 70 ng/mL) in chemo-naive patients with stage IV adenocarcinoma. cfDNA, cell-free DNA; NSCLC, non-small-cell lung cancer; PFS, progression-free survival; OS, overall survival.

Techniques Used: Concentration Assay

Circulating cfDNA time points coded by NSCLC patient identification number Graphical presentation of the association between the cfDNA level and the assessment of radiological response in patients with disease progression (A) and without progression (B) Change in the cfDNA concentration from baseline to best response, according to the radiological best response category; x-axis displays the time to clinical tumor progression. cfDNA, cell-free DNA; NSCLC, non-small-cell lung cancer; PR, partial response; SD, stable disease; PD, progression of disease * Kruskal-Wallis test among PR, SD and PD groups ** Data are expressed as the median, followed by the interquartile range in parentheses *** Wilcoxon signed rank test between the cfDNA concentration at baseline and at best response.
Figure Legend Snippet: Circulating cfDNA time points coded by NSCLC patient identification number Graphical presentation of the association between the cfDNA level and the assessment of radiological response in patients with disease progression (A) and without progression (B) Change in the cfDNA concentration from baseline to best response, according to the radiological best response category; x-axis displays the time to clinical tumor progression. cfDNA, cell-free DNA; NSCLC, non-small-cell lung cancer; PR, partial response; SD, stable disease; PD, progression of disease * Kruskal-Wallis test among PR, SD and PD groups ** Data are expressed as the median, followed by the interquartile range in parentheses *** Wilcoxon signed rank test between the cfDNA concentration at baseline and at best response.

Techniques Used: Concentration Assay

Circulating cfDNA kinetics in patients with NSCLCQuantitative cfDNA dynamics during treatment for NSCLC (A) Change in the cfDNA concentration from baseline to disease progression, according to the radiological best response category. (B) Change in the cfDNA concentration from the previous level to disease progression, according to the radiological best response category. Colors and symbols in the panel represent individual patient cfDNA kinetics; x-axis displays the time to clinical tumor progression. cfDNA, cell-free DNA; NSCLC, non-small-cell lung cancer; PR, partial response; SD, stable disease; PD, progression of disease * Kruskal-Wallis test among PR, SD and PD groups ** Data are expressed as the median, followed by the interquartile range in parentheses. *** Wilcoxon signed rank test between the cfDNA level at disease progression and the baseline or previous cfDNA level.
Figure Legend Snippet: Circulating cfDNA kinetics in patients with NSCLCQuantitative cfDNA dynamics during treatment for NSCLC (A) Change in the cfDNA concentration from baseline to disease progression, according to the radiological best response category. (B) Change in the cfDNA concentration from the previous level to disease progression, according to the radiological best response category. Colors and symbols in the panel represent individual patient cfDNA kinetics; x-axis displays the time to clinical tumor progression. cfDNA, cell-free DNA; NSCLC, non-small-cell lung cancer; PR, partial response; SD, stable disease; PD, progression of disease * Kruskal-Wallis test among PR, SD and PD groups ** Data are expressed as the median, followed by the interquartile range in parentheses. *** Wilcoxon signed rank test between the cfDNA level at disease progression and the baseline or previous cfDNA level.

Techniques Used: Concentration Assay

30) Product Images from "gbpA as a Novel qPCR Target for the Species-Specific Detection of Vibrio cholerae O1, O139, Non-O1/Non-O139 in Environmental, Stool, and Historical Continuous Plankton Recorder Samples"

Article Title: gbpA as a Novel qPCR Target for the Species-Specific Detection of Vibrio cholerae O1, O139, Non-O1/Non-O139 in Environmental, Stool, and Historical Continuous Plankton Recorder Samples

Journal: PLoS ONE

doi: 10.1371/journal.pone.0123983

Performance of the qPCR assay for detection of V . cholerae in artificially degraded DNA samples. (A) Electropherogram plot obtained by Agilent Bioanalyzer analysis of artificially fragmented genomic DNA of V . cholerae ATCC 39315. (B) Plot of mean Cq-values from three replicates tested against the V . cholerae artificially fragmented DNA inputs. Error bars indicate the standard deviations of the means.
Figure Legend Snippet: Performance of the qPCR assay for detection of V . cholerae in artificially degraded DNA samples. (A) Electropherogram plot obtained by Agilent Bioanalyzer analysis of artificially fragmented genomic DNA of V . cholerae ATCC 39315. (B) Plot of mean Cq-values from three replicates tested against the V . cholerae artificially fragmented DNA inputs. Error bars indicate the standard deviations of the means.

Techniques Used: Real-time Polymerase Chain Reaction

31) Product Images from "Bead-linked transposomes enable a normalization-free workflow for NGS library preparation"

Article Title: Bead-linked transposomes enable a normalization-free workflow for NGS library preparation

Journal: BMC Genomics

doi: 10.1186/s12864-018-5096-9

Application of Nextera DNA Flex to bacterial amplicons. a Libraries prepared using Nextera DNA Flex showed more consistent, even coverage compared with libraries prepared using Nextera XT; data depicts the sequence coverage of libraries prepared from the 3 kb E. coli amplicon. b PCR products ranging in size from 50 bp to 3 kb amplified from E. coli gDNA visualized on a 1% agarose gel. c Libraries prepared from a 1 ng input of these E. coli amplicons resulted in Bioanalyzer traces that depicted a slight increase in fragment size with increasing amplicon size. d Libraries were sequenced on a MiSeq and coverage of the E. coli genome determined for the different amplicon fragment size inputs. Sequenceable libraries were generated from amplicons ranging in size from 50 bp to 3 kb. e When sequencing data was downsampled to 25,000 reads, the larger fragment inputs were reaching a coverage maximum
Figure Legend Snippet: Application of Nextera DNA Flex to bacterial amplicons. a Libraries prepared using Nextera DNA Flex showed more consistent, even coverage compared with libraries prepared using Nextera XT; data depicts the sequence coverage of libraries prepared from the 3 kb E. coli amplicon. b PCR products ranging in size from 50 bp to 3 kb amplified from E. coli gDNA visualized on a 1% agarose gel. c Libraries prepared from a 1 ng input of these E. coli amplicons resulted in Bioanalyzer traces that depicted a slight increase in fragment size with increasing amplicon size. d Libraries were sequenced on a MiSeq and coverage of the E. coli genome determined for the different amplicon fragment size inputs. Sequenceable libraries were generated from amplicons ranging in size from 50 bp to 3 kb. e When sequencing data was downsampled to 25,000 reads, the larger fragment inputs were reaching a coverage maximum

Techniques Used: Sequencing, Amplification, Polymerase Chain Reaction, Agarose Gel Electrophoresis, Generated

Bioanalyzer traces of libraries prepared from various sample types and species. a Libraries prepared from samples with a varied degree of formalin fixation; a higher ΔCq indicates more FFPE-induced DNA degradation compared with a positive control. b Increasing FFPE-induced DNA degradation has a small effect on average fragment size but a marked effect on the total library yield. Increasing the DNA input from 100 ng to 150 ng did not increase library yield, indicating bead saturation at a DNA input of around 100 ng regardless of the degree of DNA degradation. c Libraries prepared from gDNA from a range of animal (human, Angus, and mouse), plant (Arabidopsis and alfalfa), and bacterial ( E. coli and B. cereus ) species
Figure Legend Snippet: Bioanalyzer traces of libraries prepared from various sample types and species. a Libraries prepared from samples with a varied degree of formalin fixation; a higher ΔCq indicates more FFPE-induced DNA degradation compared with a positive control. b Increasing FFPE-induced DNA degradation has a small effect on average fragment size but a marked effect on the total library yield. Increasing the DNA input from 100 ng to 150 ng did not increase library yield, indicating bead saturation at a DNA input of around 100 ng regardless of the degree of DNA degradation. c Libraries prepared from gDNA from a range of animal (human, Angus, and mouse), plant (Arabidopsis and alfalfa), and bacterial ( E. coli and B. cereus ) species

Techniques Used: Formalin-fixed Paraffin-Embedded, Positive Control

Application of Nextera DNA Flex to human amplicons. a Human leukocyte antigen (HLA) gene amplicons used as inputs for library preparation visualized on a 1% agarose gel. Lanes and expected amplicon sizes are as follows: 1, KBL Ladder; 2, HLA-A (4.1 kb); 3, HLA-B (2.8 kb); 4, HLA-C (4.2 kb); 5, HLA-DPA1 (10.3 kb); 6, HLA-DPB1 (9.7 kb); 7, HLA-DQA1 (7.3 kb); 8, HLA-DRB2 (4.6 kb); 9, HLA-DQB1 (7.1 kb). b Nextera DNA Flex library yields of all HLA amplicons were within the acceptable values of > 4 ng/μl and 9–13 ng/μl for 1 ng and 100–300 ng inputs, respectively. The yields for Nextera DNA Flex libraries were higher than for those prepared using TruSight HLA; for TruSight HLA, libraries were prepared from 1 ng of each amplicon and then pooled. c The Bioanalyzer profiles depict library fragment size distributions within the acceptable range; the distribution is narrower for the Nextera DNA Flex libraries (1 ng DNA inputs) than the TruSight HLA libraries. d Sequencing coverage depth and uniformity were higher for libraries prepared using Nextera DNA Flex (Flex) compared with TruSight HLA (TS HLA). e Libraries were sequenced on a NextSeq 550, with downsampling to 25,000 reads per amplicon. Library preparation using Nextera DNA Flex (orange) resulted in more uniform coverage of the entire human mitochondrial chromosome when compared with Nextera XT (grey). The location of the PCR primers used to create the two mtDNA amplicons are depicted by blue and red arrows. Dotted-line rectangle indicates the D-Loop region. f Zoomed in view shows more uniform coverage with Nextera DNA Flex within the D-Loop region
Figure Legend Snippet: Application of Nextera DNA Flex to human amplicons. a Human leukocyte antigen (HLA) gene amplicons used as inputs for library preparation visualized on a 1% agarose gel. Lanes and expected amplicon sizes are as follows: 1, KBL Ladder; 2, HLA-A (4.1 kb); 3, HLA-B (2.8 kb); 4, HLA-C (4.2 kb); 5, HLA-DPA1 (10.3 kb); 6, HLA-DPB1 (9.7 kb); 7, HLA-DQA1 (7.3 kb); 8, HLA-DRB2 (4.6 kb); 9, HLA-DQB1 (7.1 kb). b Nextera DNA Flex library yields of all HLA amplicons were within the acceptable values of > 4 ng/μl and 9–13 ng/μl for 1 ng and 100–300 ng inputs, respectively. The yields for Nextera DNA Flex libraries were higher than for those prepared using TruSight HLA; for TruSight HLA, libraries were prepared from 1 ng of each amplicon and then pooled. c The Bioanalyzer profiles depict library fragment size distributions within the acceptable range; the distribution is narrower for the Nextera DNA Flex libraries (1 ng DNA inputs) than the TruSight HLA libraries. d Sequencing coverage depth and uniformity were higher for libraries prepared using Nextera DNA Flex (Flex) compared with TruSight HLA (TS HLA). e Libraries were sequenced on a NextSeq 550, with downsampling to 25,000 reads per amplicon. Library preparation using Nextera DNA Flex (orange) resulted in more uniform coverage of the entire human mitochondrial chromosome when compared with Nextera XT (grey). The location of the PCR primers used to create the two mtDNA amplicons are depicted by blue and red arrows. Dotted-line rectangle indicates the D-Loop region. f Zoomed in view shows more uniform coverage with Nextera DNA Flex within the D-Loop region

Techniques Used: Agarose Gel Electrophoresis, Amplification, Sequencing, Polymerase Chain Reaction

32) Product Images from "Folate deficiency facilitates recruitment of upstream binding factor to hot spots of DNA double-strand breaks of rRNA genes and promotes its transcription"

Article Title: Folate deficiency facilitates recruitment of upstream binding factor to hot spots of DNA double-strand breaks of rRNA genes and promotes its transcription

Journal: Nucleic Acids Research

doi: 10.1093/nar/gkw1208

DSBs enrichment workflow and specificity of the DNA DSBs ( A ) DSBs enrichment workflow by MTX treatment or Restriction endonuclease digestion for quality control. Fragments released from the streptavidin beads were amplified by PCR using sequencing primers and sequenced. ( B ) Quality control of in situ digestion and blunt-ending by capillary electrophoresis. The top two traces are for the endonuclease digestion and blunt-ending performed in liquid; the bottom two traces are in low melting point agarose gel. I represents the size of digestion product of a 567-bp fluorescence-labeled DNA fragment by restriction digestion while II shows the size of digestion product after blunt-ending; III and IV represent the above reactions respectively in low melting point agarose gel. The arrow in black represents the complete blunt-ending. X-axis represents the size of fragments(bp), Y-axis represents the detector signal of peak(rfu). ( C ) DSBs enrichment products separated by agarose gel electrophoresis indicated by white box. ( D–F ) Capillary electrophoresis to detect DSB enrichment products after SbfI ( D ), PmeI ( E ) and HindIII ( F ) digestion. TA clone sequencing confirmed the results. The arrow in red indicates the DSB enrichments on Capillary electrophoresis; the circle marked with red-dotted lines shows the restriction sites; the arrow in black shows the ligation point. X-axis represents the size of fragments(bp), while Y-axis represents the detector signal of peak(rfu). ( G, H ) Capillary electrophoresis to detect DSB enrichment products of normal mESCs cultured in complete medium ( G ) and cultured in complete medium with 0.12 μM MTX ( H ). The arrow in red indicates the DSB enrichments. X-axis represents the size of fragments(bp), while Y-axis represents the detector signal of peak(rfu).
Figure Legend Snippet: DSBs enrichment workflow and specificity of the DNA DSBs ( A ) DSBs enrichment workflow by MTX treatment or Restriction endonuclease digestion for quality control. Fragments released from the streptavidin beads were amplified by PCR using sequencing primers and sequenced. ( B ) Quality control of in situ digestion and blunt-ending by capillary electrophoresis. The top two traces are for the endonuclease digestion and blunt-ending performed in liquid; the bottom two traces are in low melting point agarose gel. I represents the size of digestion product of a 567-bp fluorescence-labeled DNA fragment by restriction digestion while II shows the size of digestion product after blunt-ending; III and IV represent the above reactions respectively in low melting point agarose gel. The arrow in black represents the complete blunt-ending. X-axis represents the size of fragments(bp), Y-axis represents the detector signal of peak(rfu). ( C ) DSBs enrichment products separated by agarose gel electrophoresis indicated by white box. ( D–F ) Capillary electrophoresis to detect DSB enrichment products after SbfI ( D ), PmeI ( E ) and HindIII ( F ) digestion. TA clone sequencing confirmed the results. The arrow in red indicates the DSB enrichments on Capillary electrophoresis; the circle marked with red-dotted lines shows the restriction sites; the arrow in black shows the ligation point. X-axis represents the size of fragments(bp), while Y-axis represents the detector signal of peak(rfu). ( G, H ) Capillary electrophoresis to detect DSB enrichment products of normal mESCs cultured in complete medium ( G ) and cultured in complete medium with 0.12 μM MTX ( H ). The arrow in red indicates the DSB enrichments. X-axis represents the size of fragments(bp), while Y-axis represents the detector signal of peak(rfu).

Techniques Used: Amplification, Polymerase Chain Reaction, Sequencing, In Situ, Electrophoresis, Agarose Gel Electrophoresis, Fluorescence, Labeling, Ligation, Cell Culture

33) Product Images from "Major 5′terminally deleted enterovirus populations modulate type I IFN response in acute myocarditis patients and in human cultured cardiomyocytes"

Article Title: Major 5′terminally deleted enterovirus populations modulate type I IFN response in acute myocarditis patients and in human cultured cardiomyocytes

Journal: Scientific Reports

doi: 10.1038/s41598-020-67648-5

Quantitative detection of undeleted and 5′terminally deleted EV-B populations in clinical samples. ( A ) Electrophoregrams of RACE-PCR FL synthetic cDNA (135 bp ± 10%). ( B ) Quantification of EV-B full-length (FL) form after serial tenfold dilutions of synthetic FL form diluted in human cardiac (○) or plasmatic (∆) total RNA extracts of EV-negative human samples (n = 3). Linear regression curve between cDNA copies detected per µL and concentration of full-length synthetic cDNA. ( C ) Electrophoregrams of TD50 synthetic cDNA (55 bp ± 10%) after RACE-PCR. Image obtained from “Agilent 2,100 Bioanalyzer” after analysis with High sensitivity DNA (Agilent). ( D ) Quantification of EV-B 5′terminally deleted of 50 nt (TD50) form after serial tenfold dilutions of synthetic TD50 form diluted in human cardiac (○) or plasmatic (∆) total RNA extracts of EV-negative human samples (n = 3). Linear regression curve between cDNA copies detected per µL and concentration of TD50 form of synthetic cDNA. ( E ) Electrophoregrams of FL and TD50 mix (95% of TD50 form with 5% of FL form) synthetic cDNA after RACE-PCR. Image obtained from “Agilent 2,100 Bioanalyzer” after analysis with High sensitivity DNA (Agilent). ( F ) Quantification of EV-B FL and TD50 mix (95% of TD50 form with 5% of FL form) forms after serial tenfold dilutions of synthetic FL and TD50 synthetic forms diluted in human cardiac (○) or plasmatic (∆) total RNA extracts of EV-negative human samples (n = 3). Linear regression curve between cDNA copies detected per µL and concentration of mix FL and TD50 form of synthetic cDNA. The dashed lines indicate the thresholds of detection. Linear regression was performed, and slopes were compared using Spearman test. FL, full-length; TD, terminally deleted, bp, base paired.
Figure Legend Snippet: Quantitative detection of undeleted and 5′terminally deleted EV-B populations in clinical samples. ( A ) Electrophoregrams of RACE-PCR FL synthetic cDNA (135 bp ± 10%). ( B ) Quantification of EV-B full-length (FL) form after serial tenfold dilutions of synthetic FL form diluted in human cardiac (○) or plasmatic (∆) total RNA extracts of EV-negative human samples (n = 3). Linear regression curve between cDNA copies detected per µL and concentration of full-length synthetic cDNA. ( C ) Electrophoregrams of TD50 synthetic cDNA (55 bp ± 10%) after RACE-PCR. Image obtained from “Agilent 2,100 Bioanalyzer” after analysis with High sensitivity DNA (Agilent). ( D ) Quantification of EV-B 5′terminally deleted of 50 nt (TD50) form after serial tenfold dilutions of synthetic TD50 form diluted in human cardiac (○) or plasmatic (∆) total RNA extracts of EV-negative human samples (n = 3). Linear regression curve between cDNA copies detected per µL and concentration of TD50 form of synthetic cDNA. ( E ) Electrophoregrams of FL and TD50 mix (95% of TD50 form with 5% of FL form) synthetic cDNA after RACE-PCR. Image obtained from “Agilent 2,100 Bioanalyzer” after analysis with High sensitivity DNA (Agilent). ( F ) Quantification of EV-B FL and TD50 mix (95% of TD50 form with 5% of FL form) forms after serial tenfold dilutions of synthetic FL and TD50 synthetic forms diluted in human cardiac (○) or plasmatic (∆) total RNA extracts of EV-negative human samples (n = 3). Linear regression curve between cDNA copies detected per µL and concentration of mix FL and TD50 form of synthetic cDNA. The dashed lines indicate the thresholds of detection. Linear regression was performed, and slopes were compared using Spearman test. FL, full-length; TD, terminally deleted, bp, base paired.

Techniques Used: Polymerase Chain Reaction, Concentration Assay

34) Product Images from "Generating Exome Enriched Sequencing Libraries from Formalin-Fixed, Paraffin-Embedded Tissue DNA for Next Generation Sequencing"

Article Title: Generating Exome Enriched Sequencing Libraries from Formalin-Fixed, Paraffin-Embedded Tissue DNA for Next Generation Sequencing

Journal: Current protocols in human genetics

doi: 10.1002/cphg.27

DNA fragmentation quality check. 50ng of FFPE derived DNA was sheared using the Covaris E220 instrument. Following clean up each sample was diluted 1:5 and run on a 2100 BioAnalyzer High Sensitivity DNA chip to check the DNA fragment size distribution and sample concentration. A broad distribution of sizes should be expected between 100-2000bp is typical, with the majority of the fragments in the 200-800bp range.
Figure Legend Snippet: DNA fragmentation quality check. 50ng of FFPE derived DNA was sheared using the Covaris E220 instrument. Following clean up each sample was diluted 1:5 and run on a 2100 BioAnalyzer High Sensitivity DNA chip to check the DNA fragment size distribution and sample concentration. A broad distribution of sizes should be expected between 100-2000bp is typical, with the majority of the fragments in the 200-800bp range.

Techniques Used: Formalin-fixed Paraffin-Embedded, Derivative Assay, Chromatin Immunoprecipitation, Concentration Assay

35) Product Images from "Large-Scale Low-Cost NGS Library Preparation Using a Robust Tn5 Purification and Tagmentation Protocol"

Article Title: Large-Scale Low-Cost NGS Library Preparation Using a Robust Tn5 Purification and Tagmentation Protocol

Journal: G3: Genes|Genomes|Genetics

doi: 10.1534/g3.117.300257

NGS-library preparation using the homemade Tn5 constructs. (A) Workflow of Tn5 loading, cDNA tagmentation, and subsequent NGS library preparation for duplex index (Illumina i5/i7) full-length cDNA sequencing. Tn5 molecules are shown as gray hexamers. The double-stranded part of the linker oligonucleotide, the mosaic element, is shown in gray with a yellow circle depicting the phosphorylated 3′ end. The 5′ overhangs as templates for the i5 or i7 index adapter primers are shown in red or blue, respectively. cDNA is shown as two parallel black lines with a 3′ poly(A) tail. The synthesis of the 5′ overhang complementary strand (gap-filling step during PCR amplification) is depicted as a dotted arrow. i5 index adapter primer is shown in dark blue, while i7 index adapter primer is shown in orange. Fragments that are lost during library preparation are transparent. (B) Bioanalyzer traces of NGS libraries processed with different concentrations of the in-house-produced Tn5 R27S,E54K,L372P using only homemade or inexpensive commercially available reagents for tagmentation and subsequent PCR reaction. Following the tagmentation protocol presented here, fragmentation of the cDNA works best when using Tn5 R27S,E54K,L372P at a concentration of 30 ng/μl. (C) Heat scatter plot showing the correlation of read counts between libraries processed with either in-house-produced Tn5 E54K,L372P or Tn5 R27S,E54K,L372P . Data of three technical replicates per condition were pooled for this analysis. (D) Heat scatter plot showing the correlation of gene counts between libraries processed using either in-house-produced Tn5 R27S,E54K,L372P and the protocol presented here or the Nextera XT DNA library preparation kit following the manufacturer’s instructions. Data of three technical replicates per condition were pooled for this analysis. (E) Pairwise correlation of read counts between three technical replicates (samples processed from the same cDNA on the same day) when using homemade Tn5 R27S,E54K,L372P and the tagmentation protocol presented here. The Pearson correlation of r = 0.99 between all samples demonstrates high reproducibility of both the enzyme and the protocol. (F) Heat map analysis of gene counts in technical replicates processed from the same cDNA but on different days. The color code indicates the Pearson correlation between samples (see legend on the right side). NGS, next-generation sequencing; PCR, polymerase chain reaction; SDS, sodium dodecyl sulfate.
Figure Legend Snippet: NGS-library preparation using the homemade Tn5 constructs. (A) Workflow of Tn5 loading, cDNA tagmentation, and subsequent NGS library preparation for duplex index (Illumina i5/i7) full-length cDNA sequencing. Tn5 molecules are shown as gray hexamers. The double-stranded part of the linker oligonucleotide, the mosaic element, is shown in gray with a yellow circle depicting the phosphorylated 3′ end. The 5′ overhangs as templates for the i5 or i7 index adapter primers are shown in red or blue, respectively. cDNA is shown as two parallel black lines with a 3′ poly(A) tail. The synthesis of the 5′ overhang complementary strand (gap-filling step during PCR amplification) is depicted as a dotted arrow. i5 index adapter primer is shown in dark blue, while i7 index adapter primer is shown in orange. Fragments that are lost during library preparation are transparent. (B) Bioanalyzer traces of NGS libraries processed with different concentrations of the in-house-produced Tn5 R27S,E54K,L372P using only homemade or inexpensive commercially available reagents for tagmentation and subsequent PCR reaction. Following the tagmentation protocol presented here, fragmentation of the cDNA works best when using Tn5 R27S,E54K,L372P at a concentration of 30 ng/μl. (C) Heat scatter plot showing the correlation of read counts between libraries processed with either in-house-produced Tn5 E54K,L372P or Tn5 R27S,E54K,L372P . Data of three technical replicates per condition were pooled for this analysis. (D) Heat scatter plot showing the correlation of gene counts between libraries processed using either in-house-produced Tn5 R27S,E54K,L372P and the protocol presented here or the Nextera XT DNA library preparation kit following the manufacturer’s instructions. Data of three technical replicates per condition were pooled for this analysis. (E) Pairwise correlation of read counts between three technical replicates (samples processed from the same cDNA on the same day) when using homemade Tn5 R27S,E54K,L372P and the tagmentation protocol presented here. The Pearson correlation of r = 0.99 between all samples demonstrates high reproducibility of both the enzyme and the protocol. (F) Heat map analysis of gene counts in technical replicates processed from the same cDNA but on different days. The color code indicates the Pearson correlation between samples (see legend on the right side). NGS, next-generation sequencing; PCR, polymerase chain reaction; SDS, sodium dodecyl sulfate.

Techniques Used: Next-Generation Sequencing, Construct, Sequencing, Polymerase Chain Reaction, Amplification, Produced, Concentration Assay

36) Product Images from "Investigation of Gene Expressions of Myeloma Cells in the Bone Marrow of Multiple Myeloma Patients by Transcriptome Analysis"

Article Title: Investigation of Gene Expressions of Myeloma Cells in the Bone Marrow of Multiple Myeloma Patients by Transcriptome Analysis

Journal: Balkan Medical Journal

doi: 10.4274/balkanmedj.2018.0356

In Agilent Bioanalyzer gel-like image of cDNA. This image shows produced cDNA samples quality controls. After library construction for the quality of the libraries was validated using the Agilent High Sensitivity DNA kit on the Agilent 2100 Bioanalyzer. DNA ladder (L), Lanes 1-3-5 (control cDNA library), the lanes 3-5 are ten-fold diluted sample 1. Lanels 2-4-6 (Multiple myeloma cDNA library). The lanes 4-6 are ten- fold diluted sample 2. Lane 7 (negative). Green lines indicate the low weight (35 base pairs) DNA ladder, Purple lines the high weight (10380 base pairs) DNA ladder.
Figure Legend Snippet: In Agilent Bioanalyzer gel-like image of cDNA. This image shows produced cDNA samples quality controls. After library construction for the quality of the libraries was validated using the Agilent High Sensitivity DNA kit on the Agilent 2100 Bioanalyzer. DNA ladder (L), Lanes 1-3-5 (control cDNA library), the lanes 3-5 are ten-fold diluted sample 1. Lanels 2-4-6 (Multiple myeloma cDNA library). The lanes 4-6 are ten- fold diluted sample 2. Lane 7 (negative). Green lines indicate the low weight (35 base pairs) DNA ladder, Purple lines the high weight (10380 base pairs) DNA ladder.

Techniques Used: Produced, cDNA Library Assay

37) Product Images from "New evidence that a large proportion of human blood plasma cell-free DNA is localized in exosomes"

Article Title: New evidence that a large proportion of human blood plasma cell-free DNA is localized in exosomes

Journal: PLoS ONE

doi: 10.1371/journal.pone.0183915

Analysis of exosome DNA by agarose gel separation and Agilent Bioanalyzer. A, Exosome DNA without RNase treatment. B, Exosome DNA with RNase treatment. High molecular weight band is removed by RNase treatment indicating that band represents RNA. Low molecular weight band is resistant to RNase treatment indicating that it is DNA. Majority of exosome DNA are in 200 bp size range. C, Overlaid Agilent 2100 Bioanalyzer electropherograms. Exosome DNA was extracted from two individual donors. Exosome DNA from both donors were either treated with RNase or not treated. RNase treated and not treated DNA were analyzed by Agilent Bioanalyzer and RNase treated and not treated electropherograms were overlaid.
Figure Legend Snippet: Analysis of exosome DNA by agarose gel separation and Agilent Bioanalyzer. A, Exosome DNA without RNase treatment. B, Exosome DNA with RNase treatment. High molecular weight band is removed by RNase treatment indicating that band represents RNA. Low molecular weight band is resistant to RNase treatment indicating that it is DNA. Majority of exosome DNA are in 200 bp size range. C, Overlaid Agilent 2100 Bioanalyzer electropherograms. Exosome DNA was extracted from two individual donors. Exosome DNA from both donors were either treated with RNase or not treated. RNase treated and not treated DNA were analyzed by Agilent Bioanalyzer and RNase treated and not treated electropherograms were overlaid.

Techniques Used: Agarose Gel Electrophoresis, Molecular Weight

38) Product Images from "Automated DNA extraction using cellulose magnetic beads can improve EGFR point mutation detection with liquid biopsy by efficiently recovering short and long DNA fragments"

Article Title: Automated DNA extraction using cellulose magnetic beads can improve EGFR point mutation detection with liquid biopsy by efficiently recovering short and long DNA fragments

Journal: Oncotarget

doi: 10.18632/oncotarget.25388

Association between EGFR L858R positivity and DNA amounts in regions 1 and 2 Peripheral blood was collected from 40 patients with NSCLC who carried EGFR L858R as verified by tissue biopsy and plasma DNA extracted by method 1000-A. DNA concentration and molarity were analyzed according to plasma L858R positivity as determined by the MPB-QP method. Concentration and molality of regions 1 and 2 DNA fragments were measured by an Agilent Bioanalyzer. The concentration ( A – C ) and molarity ( D – F ) of regions 1 and 2 are shown. Statistical analyses were performed with the Mann–Whitney U test. Two asterisks denote p
Figure Legend Snippet: Association between EGFR L858R positivity and DNA amounts in regions 1 and 2 Peripheral blood was collected from 40 patients with NSCLC who carried EGFR L858R as verified by tissue biopsy and plasma DNA extracted by method 1000-A. DNA concentration and molarity were analyzed according to plasma L858R positivity as determined by the MPB-QP method. Concentration and molality of regions 1 and 2 DNA fragments were measured by an Agilent Bioanalyzer. The concentration ( A – C ) and molarity ( D – F ) of regions 1 and 2 are shown. Statistical analyses were performed with the Mann–Whitney U test. Two asterisks denote p

Techniques Used: Concentration Assay, MANN-WHITNEY

Size distribution of plasma DNA analyzed by Agilent Bioanalyzer and its difference according to extraction method ( A ) Representative size distribution pattern with each plasma DNA extraction method. Blue shows 200-M, green is 200-A, and red is 1000-A. ( B ) The definitions of “Region 1” and “Region 2”. DNA concentration and molality were measured by an Agilent Bioanalyzer. Comparison among DNA isolation procedures (200-M, 200-A, and 1000-A) was performed for concentration ( C , D ) and molarity ( E , F ) with Freidman's test and multiple pairwise comparisons.
Figure Legend Snippet: Size distribution of plasma DNA analyzed by Agilent Bioanalyzer and its difference according to extraction method ( A ) Representative size distribution pattern with each plasma DNA extraction method. Blue shows 200-M, green is 200-A, and red is 1000-A. ( B ) The definitions of “Region 1” and “Region 2”. DNA concentration and molality were measured by an Agilent Bioanalyzer. Comparison among DNA isolation procedures (200-M, 200-A, and 1000-A) was performed for concentration ( C , D ) and molarity ( E , F ) with Freidman's test and multiple pairwise comparisons.

Techniques Used: DNA Extraction, Concentration Assay

39) Product Images from "Mapping chromatin accessibility and active regulatory elements reveals new pathological mechanisms in human gliomas"

Article Title: Mapping chromatin accessibility and active regulatory elements reveals new pathological mechanisms in human gliomas

Journal: bioRxiv

doi: 10.1101/867861

A. A characteristic appearance of a single nucleosome and their multiplications in the material isolated for ATAC-seq separated on DNA Agilent chips (PA04 sample). B. Validation of immunoprecipitation efficiency with antibodies used in the experiments (for details see Methods). Chromatin immunoprecipitation (ChIP)-quantitative polymerase chain reaction (qPCR) analysis of histone modification enrichment in control regions: GAPDH promoter (active chromatin marks, H3K4me3, H3K27ac) and HOXA7 gene body (repressive mark, H3K27me3). Results are calculated as fold enrichment over negative control (immunoprecipitation with normal IgG) and represented as mean ± SD (H3K4me3 ChIP-qPCR n=6, H3K27ac ChIP-qPCR n=5, H3K27me3 ChIP-qPCR n=3n = 4). Statistical significance **P
Figure Legend Snippet: A. A characteristic appearance of a single nucleosome and their multiplications in the material isolated for ATAC-seq separated on DNA Agilent chips (PA04 sample). B. Validation of immunoprecipitation efficiency with antibodies used in the experiments (for details see Methods). Chromatin immunoprecipitation (ChIP)-quantitative polymerase chain reaction (qPCR) analysis of histone modification enrichment in control regions: GAPDH promoter (active chromatin marks, H3K4me3, H3K27ac) and HOXA7 gene body (repressive mark, H3K27me3). Results are calculated as fold enrichment over negative control (immunoprecipitation with normal IgG) and represented as mean ± SD (H3K4me3 ChIP-qPCR n=6, H3K27ac ChIP-qPCR n=5, H3K27me3 ChIP-qPCR n=3n = 4). Statistical significance **P

Techniques Used: Isolation, Immunoprecipitation, Chromatin Immunoprecipitation, Real-time Polymerase Chain Reaction, Modification, Negative Control

40) 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 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

Related Articles

Amplification:

Article Title: Quartz-Seq2: a high-throughput single-cell RNA-sequencing method that effectively uses limited sequence reads
Article Snippet: .. We checked the length distribution of amplified cDNA with an Agilent High Sensitivity DNA Kit (Agilent). .. The typical average size of the amplified cDNA in Quartz-Seq2 was approximately 1400 bp (Additional file : Figure S2c).

RNA Sequencing Assay:

Article Title: Expanded subgenomic mRNA transcriptome and coding capacity of a nidovirus
Article Snippet: .. The final dsDNA library was validated with an Agilent High Sensitivity DNA Kit (Agilent Technologies) and subjected to RNA-seq using Illumina HiSeq (paired-end reads, 100-bp read length) with one sample per lane. ..

Next-Generation Sequencing:

Article Title: Expanding Duplication of Free Fatty Acid Receptor-2 (GPR43) Genes in the Chicken Genome
Article Snippet: .. Libraries were then checked on an Agilent Technologies 2100 Bioanalyzer using the Agilent High Sensitivity DNA Kit and quantified by qPCR with the QPCR NGS Library Quantification kit (Agilent Technologies). .. After quantification, tagged cDNA libraries were pooled in equal ratios and a final qPCR check was performed postpooling.

Purification:

Article Title: Indication of Horizontal DNA Gene Transfer by Extracellular Vesicles
Article Snippet: .. One μl of purified DNA was analyzed using High Sensitivity DNA Kit on the Agilent 2100 Bioanalyzer (Agilent Technologies, Waldbronn, Germany), 10μl out of 40μl DNA of (i) and (ii) were separated on 0.66% agarose gel. .. To exclude that the EV-associated DNA was derived from apoptotic cells, EV were purified from a culture with increased number of dead cells (14% Trypan-blue positive), the DNA extracted thereof and analysed on agarose gel.

Real-time Polymerase Chain Reaction:

Article Title: Expanding Duplication of Free Fatty Acid Receptor-2 (GPR43) Genes in the Chicken Genome
Article Snippet: .. Libraries were then checked on an Agilent Technologies 2100 Bioanalyzer using the Agilent High Sensitivity DNA Kit and quantified by qPCR with the QPCR NGS Library Quantification kit (Agilent Technologies). .. After quantification, tagged cDNA libraries were pooled in equal ratios and a final qPCR check was performed postpooling.

Concentration Assay:

Article Title: Optimization of Extraction of Circulating RNAs from Plasma – Enabling Small RNA Sequencing
Article Snippet: .. A concluding Bioanalyzer 2100 run with the High Sensitivity DNA Kit (Agilent Technologies, Germany) that allows the analysis of DNA libraries regarding size, purity and concentration completed the workflow of library preparation. .. The obtained sequence libraries were subjected to the Illumina sequencing pipeline, passing through clonal cluster generation on a single-read flow cell (Illumina Inc., USA) by bridge amplification on the cBot (TruSeq SR Cluster Kit v3-cBOT-HS, Illumina Inc., USA) and 50 cycles sequencing-by-synthesis on the HiSeq2000 (Illumina Inc., USA).

MicroChIP Assay:

Article Title: Investigation of appropriate pre-analytical procedure for circulating free DNA from liquid biopsy
Article Snippet: .. We used the High Sensitivity DNA Kit® (Agilent Technologies Inc., Santa Clara, CA, USA, Product no. 5067–4626), a microchip, and analyzed the result with an Agilent 2100 Bioanalyzer® equipped with Expert 2100 software (Agilent Technologies Inc., Santa Clara, CA, USA) according to the manufacturer’s instructions. ..

Agarose Gel Electrophoresis:

Article Title: Indication of Horizontal DNA Gene Transfer by Extracellular Vesicles
Article Snippet: .. One μl of purified DNA was analyzed using High Sensitivity DNA Kit on the Agilent 2100 Bioanalyzer (Agilent Technologies, Waldbronn, Germany), 10μl out of 40μl DNA of (i) and (ii) were separated on 0.66% agarose gel. .. To exclude that the EV-associated DNA was derived from apoptotic cells, EV were purified from a culture with increased number of dead cells (14% Trypan-blue positive), the DNA extracted thereof and analysed on agarose gel.

Software:

Article Title: Investigation of appropriate pre-analytical procedure for circulating free DNA from liquid biopsy
Article Snippet: .. We used the High Sensitivity DNA Kit® (Agilent Technologies Inc., Santa Clara, CA, USA, Product no. 5067–4626), a microchip, and analyzed the result with an Agilent 2100 Bioanalyzer® equipped with Expert 2100 software (Agilent Technologies Inc., Santa Clara, CA, USA) according to the manufacturer’s instructions. ..

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  • 94
    Agilent technologies high sensitivity dna kit
    Test case dataset samples ( a ) <t>Bioanalyzer</t> analysis to assess the quality of the starting material for Rep1, Rep2_bad and Rep2_good. Plots show the <t>DNA</t> size distribution after fragmentation. X-axis shows the fragment size in base pairs (bp) and y-axis the florescence intensity proportional to DNA abundance (FU = florescence unit). The target fragment length was about 200bp (vertical blue line), but “Rep2_bad” fragment size distribution is shifted to the left towards smaller sizes. ( b ) QC diagnostic plots based on EM and GM scores for the test case samples Rep1 (left column), Rep2_bad (centre column) and Rep2_good (right column). Cross correlation profiles (top rows), RSC score (red dashed line) plot against the reference distribution of RSC values (middle row) and fingerprint plots (bottom row) for the three test case samples. Overall, they do not consistently single out Rep2_bad as the real problematic sample.
    High Sensitivity Dna Kit, supplied by Agilent technologies, used in various techniques. Bioz Stars score: 94/100, based on 75 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/high sensitivity dna kit/product/Agilent technologies
    Average 94 stars, based on 75 article reviews
    Price from $9.99 to $1999.99
    high sensitivity dna kit - by Bioz Stars, 2020-08
    94/100 stars
      Buy from Supplier

    88
    Agilent technologies high sensitivity dna bioanalyzer kit
    Schematic of the study. (A) )] were harvested by trypsinization and resuspended in medium, as indicated. Cell samples were lysed, generating crude nuclei preparations. Next, the transposition reaction was performed in the presence of Tn5 transposase, which cuts and ligates adapters at <t>DNA</t> regions of increased accessibility. The quality of the transposed DNA fragment libraries was assessed using a <t>Bioanalyzer.</t> Libraries were sequenced, mapped to the genome, and processed bioinformatically, and accessible genomic regions were detected (peak calling). (B) Single-cell (SC) RNA-seq [gel bead-in-emulsion (GEM) Drop-seq] workflow. Following treatment with GnRH or vehicle, cultured LβT2 cells were harvested by trypsinization and resuspended in a PBS BSA-containing buffer. Cell samples were loaded on a microfluidic chip, combined with reagents and barcoded gel beads to form Gel beads in Emulsion (GEMs), and then mixed with oil. The resulting emulsions were collected and reverse transcribed in SC droplets. Oil was removed, and barcoded cDNA was amplified, purified, and subject to quality control (QC) assessment. Amplified cDNA was incorporated into libraries, which were subsequently subject to QC evaluation using Kapa, Bioanalyzer, and Qubit. Libraries were pooled for sequencing and demultiplexed for subsequent analyses.
    High Sensitivity Dna Bioanalyzer Kit, supplied by Agilent technologies, used in various techniques. Bioz Stars score: 88/100, based on 12 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/high sensitivity dna bioanalyzer kit/product/Agilent technologies
    Average 88 stars, based on 12 article reviews
    Price from $9.99 to $1999.99
    high sensitivity dna bioanalyzer kit - by Bioz Stars, 2020-08
    88/100 stars
      Buy from Supplier

    Image Search Results


    Test case dataset samples ( a ) Bioanalyzer analysis to assess the quality of the starting material for Rep1, Rep2_bad and Rep2_good. Plots show the DNA size distribution after fragmentation. X-axis shows the fragment size in base pairs (bp) and y-axis the florescence intensity proportional to DNA abundance (FU = florescence unit). The target fragment length was about 200bp (vertical blue line), but “Rep2_bad” fragment size distribution is shifted to the left towards smaller sizes. ( b ) QC diagnostic plots based on EM and GM scores for the test case samples Rep1 (left column), Rep2_bad (centre column) and Rep2_good (right column). Cross correlation profiles (top rows), RSC score (red dashed line) plot against the reference distribution of RSC values (middle row) and fingerprint plots (bottom row) for the three test case samples. Overall, they do not consistently single out Rep2_bad as the real problematic sample.

    Journal: bioRxiv

    Article Title: A ChIC solution for ChIP-seq quality assessment

    doi: 10.1101/2020.05.19.103887

    Figure Lengend Snippet: Test case dataset samples ( a ) Bioanalyzer analysis to assess the quality of the starting material for Rep1, Rep2_bad and Rep2_good. Plots show the DNA size distribution after fragmentation. X-axis shows the fragment size in base pairs (bp) and y-axis the florescence intensity proportional to DNA abundance (FU = florescence unit). The target fragment length was about 200bp (vertical blue line), but “Rep2_bad” fragment size distribution is shifted to the left towards smaller sizes. ( b ) QC diagnostic plots based on EM and GM scores for the test case samples Rep1 (left column), Rep2_bad (centre column) and Rep2_good (right column). Cross correlation profiles (top rows), RSC score (red dashed line) plot against the reference distribution of RSC values (middle row) and fingerprint plots (bottom row) for the three test case samples. Overall, they do not consistently single out Rep2_bad as the real problematic sample.

    Article Snippet: Libraries were created using Biomek FX automatic liquid handler (Beckman Coulter), then qualitatively and quantitatively checked using Agilent High Sensitivity DNA Kit (Agilent Technologies, 5067-4627) on a Bioanalyzer 2100 (Agilent Technologies).

    Techniques: Diagnostic Assay

    A. A characteristic appearance of a single nucleosome and their multiplications in the material isolated for ATAC-seq separated on DNA Agilent chips (PA04 sample). B. Validation of immunoprecipitation efficiency with antibodies used in the experiments (for details see Methods). Chromatin immunoprecipitation (ChIP)-quantitative polymerase chain reaction (qPCR) analysis of histone modification enrichment in control regions: GAPDH promoter (active chromatin marks, H3K4me3, H3K27ac) and HOXA7 gene body (repressive mark, H3K27me3). Results are calculated as fold enrichment over negative control (immunoprecipitation with normal IgG) and represented as mean ± SD (H3K4me3 ChIP-qPCR n=6, H3K27ac ChIP-qPCR n=5, H3K27me3 ChIP-qPCR n=3n = 4). Statistical significance **P

    Journal: bioRxiv

    Article Title: Mapping chromatin accessibility and active regulatory elements reveals new pathological mechanisms in human gliomas

    doi: 10.1101/867861

    Figure Lengend Snippet: A. A characteristic appearance of a single nucleosome and their multiplications in the material isolated for ATAC-seq separated on DNA Agilent chips (PA04 sample). B. Validation of immunoprecipitation efficiency with antibodies used in the experiments (for details see Methods). Chromatin immunoprecipitation (ChIP)-quantitative polymerase chain reaction (qPCR) analysis of histone modification enrichment in control regions: GAPDH promoter (active chromatin marks, H3K4me3, H3K27ac) and HOXA7 gene body (repressive mark, H3K27me3). Results are calculated as fold enrichment over negative control (immunoprecipitation with normal IgG) and represented as mean ± SD (H3K4me3 ChIP-qPCR n=6, H3K27ac ChIP-qPCR n=5, H3K27me3 ChIP-qPCR n=3n = 4). Statistical significance **P

    Article Snippet: Also a size of DNA fragments was analyzed using the High Sensitivity DNA Kit on a 2100 Bioanalyzer (Agilent Technologies, Inc.).

    Techniques: Isolation, Immunoprecipitation, Chromatin Immunoprecipitation, Real-time Polymerase Chain Reaction, Modification, Negative Control

    Amplification and cloning of sg mRNA leader–body junctions generated from additional functional TRSs located in the genomic region between identified TRS5 and TRS6. ( A ) Diagram indicating the positions of the primers used and the estimated size for the amplified leader–body junction sequences (thick black line). The white open box represents ORF5, and the short black box represents the leader sequence in transcribed sg mRNAs. ( B ) MA104 cells were either mock-infected (M) or infected with SHFVic at an MOI of 1. At 24 hpi, total intracellular RNA was extracted and subjected to RT-PCR, and the products were separated on a 2% DNA gel. The band with the size estimated for the leader–body junction in sg mRNA5 produced from the known TRS5 is indicated by an arrow. PCR bands with sizes estimated for the leader–body junctions of ∼1.7-kb sg mRNAs are indicated by a bracket. L, ladder. ( C ) The bracketed region of the gel was excised, and the DNA was extracted and cloned into a TA vector. Forty colonies were randomly selected and subjected to restriction digestion, and the inserts were separated by gel electrophoresis. The results from 10 representative clones are shown. L, ladder. ( D ) Diagram showing the locations of the known and previously unreported body TRSs. The TRSs are indicated by black vertical bars. The previously unreported functional TRSs are within a dotted line box. The ORFs encoded by the individual sg mRNAs are indicated by white open boxes. 5-C, ORF5-C-68aa; L, leader region.

    Journal: Proceedings of the National Academy of Sciences of the United States of America

    Article Title: Expanded subgenomic mRNA transcriptome and coding capacity of a nidovirus

    doi: 10.1073/pnas.1706696114

    Figure Lengend Snippet: Amplification and cloning of sg mRNA leader–body junctions generated from additional functional TRSs located in the genomic region between identified TRS5 and TRS6. ( A ) Diagram indicating the positions of the primers used and the estimated size for the amplified leader–body junction sequences (thick black line). The white open box represents ORF5, and the short black box represents the leader sequence in transcribed sg mRNAs. ( B ) MA104 cells were either mock-infected (M) or infected with SHFVic at an MOI of 1. At 24 hpi, total intracellular RNA was extracted and subjected to RT-PCR, and the products were separated on a 2% DNA gel. The band with the size estimated for the leader–body junction in sg mRNA5 produced from the known TRS5 is indicated by an arrow. PCR bands with sizes estimated for the leader–body junctions of ∼1.7-kb sg mRNAs are indicated by a bracket. L, ladder. ( C ) The bracketed region of the gel was excised, and the DNA was extracted and cloned into a TA vector. Forty colonies were randomly selected and subjected to restriction digestion, and the inserts were separated by gel electrophoresis. The results from 10 representative clones are shown. L, ladder. ( D ) Diagram showing the locations of the known and previously unreported body TRSs. The TRSs are indicated by black vertical bars. The previously unreported functional TRSs are within a dotted line box. The ORFs encoded by the individual sg mRNAs are indicated by white open boxes. 5-C, ORF5-C-68aa; L, leader region.

    Article Snippet: The final dsDNA library was validated with an Agilent High Sensitivity DNA Kit (Agilent Technologies) and subjected to RNA-seq using Illumina HiSeq (paired-end reads, 100-bp read length) with one sample per lane.

    Techniques: Amplification, Clone Assay, Generated, Functional Assay, Sequencing, Infection, Reverse Transcription Polymerase Chain Reaction, Produced, Polymerase Chain Reaction, Plasmid Preparation, Nucleic Acid Electrophoresis

    Schematic of the study. (A) )] were harvested by trypsinization and resuspended in medium, as indicated. Cell samples were lysed, generating crude nuclei preparations. Next, the transposition reaction was performed in the presence of Tn5 transposase, which cuts and ligates adapters at DNA regions of increased accessibility. The quality of the transposed DNA fragment libraries was assessed using a Bioanalyzer. Libraries were sequenced, mapped to the genome, and processed bioinformatically, and accessible genomic regions were detected (peak calling). (B) Single-cell (SC) RNA-seq [gel bead-in-emulsion (GEM) Drop-seq] workflow. Following treatment with GnRH or vehicle, cultured LβT2 cells were harvested by trypsinization and resuspended in a PBS BSA-containing buffer. Cell samples were loaded on a microfluidic chip, combined with reagents and barcoded gel beads to form Gel beads in Emulsion (GEMs), and then mixed with oil. The resulting emulsions were collected and reverse transcribed in SC droplets. Oil was removed, and barcoded cDNA was amplified, purified, and subject to quality control (QC) assessment. Amplified cDNA was incorporated into libraries, which were subsequently subject to QC evaluation using Kapa, Bioanalyzer, and Qubit. Libraries were pooled for sequencing and demultiplexed for subsequent analyses.

    Journal: Frontiers in Endocrinology

    Article Title: Regulatory Architecture of the LβT2 Gonadotrope Cell Underlying the Response to Gonadotropin-Releasing Hormone

    doi: 10.3389/fendo.2018.00034

    Figure Lengend Snippet: Schematic of the study. (A) )] were harvested by trypsinization and resuspended in medium, as indicated. Cell samples were lysed, generating crude nuclei preparations. Next, the transposition reaction was performed in the presence of Tn5 transposase, which cuts and ligates adapters at DNA regions of increased accessibility. The quality of the transposed DNA fragment libraries was assessed using a Bioanalyzer. Libraries were sequenced, mapped to the genome, and processed bioinformatically, and accessible genomic regions were detected (peak calling). (B) Single-cell (SC) RNA-seq [gel bead-in-emulsion (GEM) Drop-seq] workflow. Following treatment with GnRH or vehicle, cultured LβT2 cells were harvested by trypsinization and resuspended in a PBS BSA-containing buffer. Cell samples were loaded on a microfluidic chip, combined with reagents and barcoded gel beads to form Gel beads in Emulsion (GEMs), and then mixed with oil. The resulting emulsions were collected and reverse transcribed in SC droplets. Oil was removed, and barcoded cDNA was amplified, purified, and subject to quality control (QC) assessment. Amplified cDNA was incorporated into libraries, which were subsequently subject to QC evaluation using Kapa, Bioanalyzer, and Qubit. Libraries were pooled for sequencing and demultiplexed for subsequent analyses.

    Article Snippet: Library QC and quantification were assessed using Nanodrop, Qubit (fluorometric quantitation, ThermoFisher Scientific), Kapa (quantification, Kapa Biosystems), High-Sensitivity DNA Bioanalyzer kit (Agilent), and qPCR of selected genes.

    Techniques: RNA Sequencing Assay, Cell Culture, Chromatin Immunoprecipitation, Amplification, Purification, Sequencing