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Illumina Inc pcr amplified
iCLIP identifies the TIA1 and TIAL1 crosslink sites with nucleotide resolution. Autoradiogram of 32 P-labelled RNA crosslinked to TIA1 (A) or TIAL1 (B) in HeLa cells. Immunoprecipitation was performed with either anti-TIA1 or anti-TIAL1 antibody using lysate from UV-crosslinked HeLa cells, cells transfected with TIA1 or TIAL1, TIA1/TIAL1 KD cells, or non-crosslinked cells. High and low RNase concentrations were used and protein G beads were used as a control. The Western blots below the autoradiograms show the input lysate used for each immunoprecipitation. (C) TIA1 and TIAL1 crosslink to uridine tracts downstream of the alternative 5′ splice sites in the CLIP4 gene. The <t>cDNA</t> positions are colour-coded for three replicate TIA1 and TIAL1 experiments, and the random barcode (shown on the left) is used to identify unique iCLIP cDNAs (number in brackets indicates the number of corresponding <t>PCR</t> duplicates). Below, the bar graphs show the cDNA count (number of cDNAs at each crosslink site). Pre-mRNA sequence is shown below with crosslink nucleotides in red. The exon and intron positions of the two isoforms of CLIP4 mRNA are shown at the bottom.
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1) Product Images from "iCLIP Predicts the Dual Splicing Effects of TIA-RNA Interactions"

Article Title: iCLIP Predicts the Dual Splicing Effects of TIA-RNA Interactions

Journal: PLoS Biology

doi: 10.1371/journal.pbio.1000530

iCLIP identifies the TIA1 and TIAL1 crosslink sites with nucleotide resolution. Autoradiogram of 32 P-labelled RNA crosslinked to TIA1 (A) or TIAL1 (B) in HeLa cells. Immunoprecipitation was performed with either anti-TIA1 or anti-TIAL1 antibody using lysate from UV-crosslinked HeLa cells, cells transfected with TIA1 or TIAL1, TIA1/TIAL1 KD cells, or non-crosslinked cells. High and low RNase concentrations were used and protein G beads were used as a control. The Western blots below the autoradiograms show the input lysate used for each immunoprecipitation. (C) TIA1 and TIAL1 crosslink to uridine tracts downstream of the alternative 5′ splice sites in the CLIP4 gene. The cDNA positions are colour-coded for three replicate TIA1 and TIAL1 experiments, and the random barcode (shown on the left) is used to identify unique iCLIP cDNAs (number in brackets indicates the number of corresponding PCR duplicates). Below, the bar graphs show the cDNA count (number of cDNAs at each crosslink site). Pre-mRNA sequence is shown below with crosslink nucleotides in red. The exon and intron positions of the two isoforms of CLIP4 mRNA are shown at the bottom.
Figure Legend Snippet: iCLIP identifies the TIA1 and TIAL1 crosslink sites with nucleotide resolution. Autoradiogram of 32 P-labelled RNA crosslinked to TIA1 (A) or TIAL1 (B) in HeLa cells. Immunoprecipitation was performed with either anti-TIA1 or anti-TIAL1 antibody using lysate from UV-crosslinked HeLa cells, cells transfected with TIA1 or TIAL1, TIA1/TIAL1 KD cells, or non-crosslinked cells. High and low RNase concentrations were used and protein G beads were used as a control. The Western blots below the autoradiograms show the input lysate used for each immunoprecipitation. (C) TIA1 and TIAL1 crosslink to uridine tracts downstream of the alternative 5′ splice sites in the CLIP4 gene. The cDNA positions are colour-coded for three replicate TIA1 and TIAL1 experiments, and the random barcode (shown on the left) is used to identify unique iCLIP cDNAs (number in brackets indicates the number of corresponding PCR duplicates). Below, the bar graphs show the cDNA count (number of cDNAs at each crosslink site). Pre-mRNA sequence is shown below with crosslink nucleotides in red. The exon and intron positions of the two isoforms of CLIP4 mRNA are shown at the bottom.

Techniques Used: Immunoprecipitation, Transfection, Western Blot, Polymerase Chain Reaction, Sequencing

2) Product Images from "Ribosome maturation by the endoribonuclease YbeY stabilizes a type 3 secretion system transcript required for virulence of enterohemorrhagic Escherichia coli"

Article Title: Ribosome maturation by the endoribonuclease YbeY stabilizes a type 3 secretion system transcript required for virulence of enterohemorrhagic Escherichia coli

Journal: The Journal of Biological Chemistry

doi: 10.1074/jbc.RA117.000300

Translation stabilizes the espD transcript. A , Northern blot analysis of espD ( left panel ) and recA ( right panel ) in WT ZAP193, Δ ybeY , and the ybeY -HTF strain backgrounds. B , transcription and translation of the first gene in the LEE4 polycistronic operon, sepL , was measured using GFP fusions. Left , the transcriptional sepL -GFP fusion (pCDR8) containing the native promoter and 8 nt of the sepL 5′-UTR was measured in ZAP193 and Δ ybeY backgrounds under T3S-permissive conditions. Right , the translational sepL -GFP fusion (pDW6) containing the native promoter and entire sepL CDS fused to GFP was similarly measured under T3S-permissive conditions. Error bars represent S.E. from biological triplicates. AU , arbitrary units. C , Western blotting for the T3S needle tip protein (EspD) in whole cells grown with and without a subinhibitory concentration of the translation initiation inhibitor kasugamycin (50 μg·ml −1 ). RecA was used as a loading control ( bottom panel ). D , RNA stability assay of espD and recA mRNAs with and without translating ribosomes. Transcription was blocked in EHEC str. ZAP193 by adding 1 mg·ml −1 rifampicin ( Rif ) ( closed circles ) or rifampicin and 1 mg·ml −1 kasugamycin ( Kas ) ( open circles ) at time 0. The relative abundance of mRNAs was monitored using quantitative RT-PCR. Error bars represent S.E. from biological triplicates. E , binding profiles for RNase E ( dark blue ), Hfq ( blue ), YbeY ( dark green ), and controls ( green ) are shown for transcripts from the LEE (ORFs are in gray ; espD is highlighted in red ). Protein binding to positive ( top ) and negative strands ( bottom ) of the transcriptome are shown. The polycistronic LEE1–5 transcripts are indicated above and below relevant ORFs.
Figure Legend Snippet: Translation stabilizes the espD transcript. A , Northern blot analysis of espD ( left panel ) and recA ( right panel ) in WT ZAP193, Δ ybeY , and the ybeY -HTF strain backgrounds. B , transcription and translation of the first gene in the LEE4 polycistronic operon, sepL , was measured using GFP fusions. Left , the transcriptional sepL -GFP fusion (pCDR8) containing the native promoter and 8 nt of the sepL 5′-UTR was measured in ZAP193 and Δ ybeY backgrounds under T3S-permissive conditions. Right , the translational sepL -GFP fusion (pDW6) containing the native promoter and entire sepL CDS fused to GFP was similarly measured under T3S-permissive conditions. Error bars represent S.E. from biological triplicates. AU , arbitrary units. C , Western blotting for the T3S needle tip protein (EspD) in whole cells grown with and without a subinhibitory concentration of the translation initiation inhibitor kasugamycin (50 μg·ml −1 ). RecA was used as a loading control ( bottom panel ). D , RNA stability assay of espD and recA mRNAs with and without translating ribosomes. Transcription was blocked in EHEC str. ZAP193 by adding 1 mg·ml −1 rifampicin ( Rif ) ( closed circles ) or rifampicin and 1 mg·ml −1 kasugamycin ( Kas ) ( open circles ) at time 0. The relative abundance of mRNAs was monitored using quantitative RT-PCR. Error bars represent S.E. from biological triplicates. E , binding profiles for RNase E ( dark blue ), Hfq ( blue ), YbeY ( dark green ), and controls ( green ) are shown for transcripts from the LEE (ORFs are in gray ; espD is highlighted in red ). Protein binding to positive ( top ) and negative strands ( bottom ) of the transcriptome are shown. The polycistronic LEE1–5 transcripts are indicated above and below relevant ORFs.

Techniques Used: Northern Blot, Western Blot, Concentration Assay, Stability Assay, Quantitative RT-PCR, Binding Assay, Protein Binding

3) Product Images from "RIP-seq analysis of eukaryotic Sm proteins identifies three major categories of Sm-containing ribonucleoproteins"

Article Title: RIP-seq analysis of eukaryotic Sm proteins identifies three major categories of Sm-containing ribonucleoproteins

Journal: Genome Biology

doi: 10.1186/gb-2014-15-1-r7

Sm proteins associate with mature mRNAs. (a) Meta-gene analysis of read density around splice sites for all Drosophila and human Sm-associated intron-containing mRNAs in all RIP-seq experiments. (b) Meta-gene analysis of read density along the gene length for all Drosophila Sm-associated mRNAs quantified from oligodT and random hexamer primed libraries. (c) Example tracks for read density along the gene length for oligodT and random hexamer primed libraries. (d) Poly(A) tail length Sm-associated mRNAs (CG3997, CG1349 and CG3776) and non-associated mRNA (RpS2) from Y12 IP in S2 cells. IN, input total RNA; IP, immunoprecipitated RNA. The labels denote the length of poly(A) tails. Oligo(dT) 20 was used as the reverse primer for the reverse transcription and subsequent PCR, therefore producing the ‘smear’ of poly(A) tail. See Figure S11 in Additional file 1 for analysis of poly(A) containing reads for selected Sm-associated mRNAs.
Figure Legend Snippet: Sm proteins associate with mature mRNAs. (a) Meta-gene analysis of read density around splice sites for all Drosophila and human Sm-associated intron-containing mRNAs in all RIP-seq experiments. (b) Meta-gene analysis of read density along the gene length for all Drosophila Sm-associated mRNAs quantified from oligodT and random hexamer primed libraries. (c) Example tracks for read density along the gene length for oligodT and random hexamer primed libraries. (d) Poly(A) tail length Sm-associated mRNAs (CG3997, CG1349 and CG3776) and non-associated mRNA (RpS2) from Y12 IP in S2 cells. IN, input total RNA; IP, immunoprecipitated RNA. The labels denote the length of poly(A) tails. Oligo(dT) 20 was used as the reverse primer for the reverse transcription and subsequent PCR, therefore producing the ‘smear’ of poly(A) tail. See Figure S11 in Additional file 1 for analysis of poly(A) containing reads for selected Sm-associated mRNAs.

Techniques Used: Random Hexamer Labeling, Immunoprecipitation, Polymerase Chain Reaction

RNA-Sm association is cell type-specific and not due to re-assortment. (a) RIP-qRT-PCR in da-Gal4 VFP-SmD1 fly ovary (anti-GFP) and S2 cells (Y12). Negative controls (Ctrl) used are 5S rRNA, Act5C and Smt3. CG9042 (Gapdh) is used as the normalization standard. snRNAs are shown separately due to the difference in scale. (b) mRNAs associated with Sm proteins in ovaries but not in S2 cells are expressed in S2 cells. t -Test for significance between IP and Ctrl: * P
Figure Legend Snippet: RNA-Sm association is cell type-specific and not due to re-assortment. (a) RIP-qRT-PCR in da-Gal4 VFP-SmD1 fly ovary (anti-GFP) and S2 cells (Y12). Negative controls (Ctrl) used are 5S rRNA, Act5C and Smt3. CG9042 (Gapdh) is used as the normalization standard. snRNAs are shown separately due to the difference in scale. (b) mRNAs associated with Sm proteins in ovaries but not in S2 cells are expressed in S2 cells. t -Test for significance between IP and Ctrl: * P

Techniques Used: Quantitative RT-PCR

U1 snRNP binds mature mRNAs. (a) Putative base pairs between the 5′ end of U1 snRNA and the CG3776 mRNA coding region (upper panel). Within the putative region of base pairing, three translationally silent point mutations were introduced (bold blue letters) to disrupt the helix (lower panel). (b) Cartoon of the S2 cell transfection construct, showing the CG3776 expression unit. CG3776endo and CG3776tag indicate locations of primers for qRT-PCR. CG3776endo amplifies both endogenous and transfected CG3776 mRNAs, whereas CG3776tag amplifies transfected CG3776 mRNA only. The black star indicates the location of the putative U1 binding site. (c) pAW vector, pAW-CG3776wt and pAW-CG3776mut were transfected into S2 cells, and CG3776wt and CG3776mut expression was measured using qRT-PCR with the CG3776endo primer pair. GAPDH was used as normalization standard. (d) After pAW-CG3776wt and pAW-CG3776mut were transfected, anti-Sm (Y12) IPs were performed using S2 cell lysate. GAPDH was used as normalization standard. (e) Proposed model of snRNP-mRNA interactions. Distinct snRNPs (U1 and potentially others) associate with mature mRNAs via base pairing and/or protein-mediated interaction. Such interactions could serve as a platform to recruit RNA processing factors that act on multiple levels of RNA metabolism. t -Test for significance between IP and control (Ctrl): * P
Figure Legend Snippet: U1 snRNP binds mature mRNAs. (a) Putative base pairs between the 5′ end of U1 snRNA and the CG3776 mRNA coding region (upper panel). Within the putative region of base pairing, three translationally silent point mutations were introduced (bold blue letters) to disrupt the helix (lower panel). (b) Cartoon of the S2 cell transfection construct, showing the CG3776 expression unit. CG3776endo and CG3776tag indicate locations of primers for qRT-PCR. CG3776endo amplifies both endogenous and transfected CG3776 mRNAs, whereas CG3776tag amplifies transfected CG3776 mRNA only. The black star indicates the location of the putative U1 binding site. (c) pAW vector, pAW-CG3776wt and pAW-CG3776mut were transfected into S2 cells, and CG3776wt and CG3776mut expression was measured using qRT-PCR with the CG3776endo primer pair. GAPDH was used as normalization standard. (d) After pAW-CG3776wt and pAW-CG3776mut were transfected, anti-Sm (Y12) IPs were performed using S2 cell lysate. GAPDH was used as normalization standard. (e) Proposed model of snRNP-mRNA interactions. Distinct snRNPs (U1 and potentially others) associate with mature mRNAs via base pairing and/or protein-mediated interaction. Such interactions could serve as a platform to recruit RNA processing factors that act on multiple levels of RNA metabolism. t -Test for significance between IP and control (Ctrl): * P

Techniques Used: Transfection, Construct, Expressing, Quantitative RT-PCR, Binding Assay, Plasmid Preparation, Activated Clotting Time Assay

4) Product Images from "An ultraprocessive, accurate reverse transcriptase encoded by a metazoan group II intron"

Article Title: An ultraprocessive, accurate reverse transcriptase encoded by a metazoan group II intron

Journal: RNA

doi: 10.1261/rna.063479.117

Error rate of various reverse transcriptases including MarathonRT, SSIV, and TGIRT. ( A ) Single-molecule sequencing method. The schematic diagram of primers used for RT and second-strand synthesis is shown on the top . The principle underlying single-molecule sequencing is shown on the bottom . Only errors that are consistent in all sequencing reads that share the same product barcode are considered as RT errors (red stars). Errors that are inconsistent among reads sharing the same product barcode (green stars) will have originated from the PCR amplification or sequencing platform. ( B ) Error rate determination for different reverse transcriptases. The table summarizing the sequencing data is shown at left . In this table, nucleotides/product (row 4) is the number of nucleotides in each RT product that are used for final analysis, after the low quality bases at the ends were trimmed. Total nucleotides (row 5) is the total number of nucleotides involved in the analysis. The total number of reads (row 2) is the raw number of sequencing reads in either forward (R1) or reverse (R2) direction for each polymerase. The unique product (row 3) is a set of sequencing reads that share the same UMI (unique molecular identifier), and only unique products that have no less than three reads were included in the analysis (row 4). Nucleotide/product (row 5) shows the number of nucleotides that are incorporated by each polymerase after trimming the primer region and low-quality nucleotides at the end. Total nucleotides (row 6) is calculated by multiplying nucleotide/product (row 5) with the number of unique products (row 4), which is the total number of nucleotides analyzed. Substitution frequency (row 7) was calculated by dividing the number of total nucleotides (row 6) by the number of mutated nucleotides. Indel (insertion–deletion) frequency (row 8) was calculated by dividing the number of unique products by the number of indel events. N.A. suggests that current sequencing depth is not able to detect indels (insertion–deletion). The bar plot showing the substitutional frequency for MarathonRT, SSIV, and TGIRT is shown on the right .
Figure Legend Snippet: Error rate of various reverse transcriptases including MarathonRT, SSIV, and TGIRT. ( A ) Single-molecule sequencing method. The schematic diagram of primers used for RT and second-strand synthesis is shown on the top . The principle underlying single-molecule sequencing is shown on the bottom . Only errors that are consistent in all sequencing reads that share the same product barcode are considered as RT errors (red stars). Errors that are inconsistent among reads sharing the same product barcode (green stars) will have originated from the PCR amplification or sequencing platform. ( B ) Error rate determination for different reverse transcriptases. The table summarizing the sequencing data is shown at left . In this table, nucleotides/product (row 4) is the number of nucleotides in each RT product that are used for final analysis, after the low quality bases at the ends were trimmed. Total nucleotides (row 5) is the total number of nucleotides involved in the analysis. The total number of reads (row 2) is the raw number of sequencing reads in either forward (R1) or reverse (R2) direction for each polymerase. The unique product (row 3) is a set of sequencing reads that share the same UMI (unique molecular identifier), and only unique products that have no less than three reads were included in the analysis (row 4). Nucleotide/product (row 5) shows the number of nucleotides that are incorporated by each polymerase after trimming the primer region and low-quality nucleotides at the end. Total nucleotides (row 6) is calculated by multiplying nucleotide/product (row 5) with the number of unique products (row 4), which is the total number of nucleotides analyzed. Substitution frequency (row 7) was calculated by dividing the number of total nucleotides (row 6) by the number of mutated nucleotides. Indel (insertion–deletion) frequency (row 8) was calculated by dividing the number of unique products by the number of indel events. N.A. suggests that current sequencing depth is not able to detect indels (insertion–deletion). The bar plot showing the substitutional frequency for MarathonRT, SSIV, and TGIRT is shown on the right .

Techniques Used: Sequencing, Polymerase Chain Reaction, Amplification

5) Product Images from "Detection of bacterial DNA from central venous catheter removed from patients by next generation sequencing: a preliminary clinical study"

Article Title: Detection of bacterial DNA from central venous catheter removed from patients by next generation sequencing: a preliminary clinical study

Journal: Annals of Clinical Microbiology and Antimicrobials

doi: 10.1186/s12941-018-0297-2

Representative agarose-gel images. a A representative agarose-gel image of PCR-amplified 16S rDNA. The number of specimens regarded as positive are shown in bold. PCR reaction of the negative control sample was performed without any DNA extracted from catheter specimen. The bands around 100 to 200 bp are considered to be primer dimers. b A representative agarose-gel image of index PCR products. The minor band, presumably due to nonspecific amplification, is indicated by an arrowhead
Figure Legend Snippet: Representative agarose-gel images. a A representative agarose-gel image of PCR-amplified 16S rDNA. The number of specimens regarded as positive are shown in bold. PCR reaction of the negative control sample was performed without any DNA extracted from catheter specimen. The bands around 100 to 200 bp are considered to be primer dimers. b A representative agarose-gel image of index PCR products. The minor band, presumably due to nonspecific amplification, is indicated by an arrowhead

Techniques Used: Agarose Gel Electrophoresis, Polymerase Chain Reaction, Amplification, Negative Control

6) Product Images from "Next-generation DNA sequencing-based assay for measuring allelic expression imbalance (AEI) of candidate neuropsychiatric disorder genes in human brain"

Article Title: Next-generation DNA sequencing-based assay for measuring allelic expression imbalance (AEI) of candidate neuropsychiatric disorder genes in human brain

Journal: BMC Genomics

doi: 10.1186/1471-2164-12-518

Reproducibility of AEI assays ( GNB1L ) . (a) [Left] Comparison of the results of independent measurements of GNB1L AEI. (Each assay was performed independently, from the isolation of brain RNA through DNA sequencing.); [Right] Regression analysis. (b) Histogram of the distribution of r 2 values obtained in regression analysis of independent AEI assays for 40 candidate genes for which replica assays were carried out. (All data are shown). (c) [Left] Comparison of the results of independent measurements of GNB1L AEI using PCR/DNA sequencing-based (top) and PCR/SNaPShot ® -based (bottom) assays. [Right] Regression analysis. [Note: the Illumina-2 AEI ratios in (a) were matched with those obtained in the Illumina-1 AEI assay, which contained fewer samples. The full set of data from the Illumina-2 assay is shown in (c).]
Figure Legend Snippet: Reproducibility of AEI assays ( GNB1L ) . (a) [Left] Comparison of the results of independent measurements of GNB1L AEI. (Each assay was performed independently, from the isolation of brain RNA through DNA sequencing.); [Right] Regression analysis. (b) Histogram of the distribution of r 2 values obtained in regression analysis of independent AEI assays for 40 candidate genes for which replica assays were carried out. (All data are shown). (c) [Left] Comparison of the results of independent measurements of GNB1L AEI using PCR/DNA sequencing-based (top) and PCR/SNaPShot ® -based (bottom) assays. [Right] Regression analysis. [Note: the Illumina-2 AEI ratios in (a) were matched with those obtained in the Illumina-1 AEI assay, which contained fewer samples. The full set of data from the Illumina-2 assay is shown in (c).]

Techniques Used: Isolation, DNA Sequencing, Polymerase Chain Reaction

7) Product Images from "Influence of Heat Events on the Composition of Airborne Bacterial Communities in Urban Ecosystems"

Article Title: Influence of Heat Events on the Composition of Airborne Bacterial Communities in Urban Ecosystems

Journal: International Journal of Environmental Research and Public Health

doi: 10.3390/ijerph15102295

Airborne bacterial 16S rRNA gene copies as determined by quantitative real-time PCR beginning and end the heat event at four selected sampling sites in Hangzhou.
Figure Legend Snippet: Airborne bacterial 16S rRNA gene copies as determined by quantitative real-time PCR beginning and end the heat event at four selected sampling sites in Hangzhou.

Techniques Used: Real-time Polymerase Chain Reaction, Sampling

8) Product Images from "Partial bisulfite conversion for unique template sequencing"

Article Title: Partial bisulfite conversion for unique template sequencing

Journal: Nucleic Acids Research

doi: 10.1093/nar/gkx1054

muSeq applied to a genomic representation. We demonstrate the application of muSeq to a genomic representation by tracing two fragments (X and Y) from a single genomic locus through the molecular and informatics processing steps. The full experiment comprises 160,000 genomic loci and about 100 fragments per locus. ( A ) We first generate a representation from genomic DNA using PstI. ( B ) The ends of restriction fragments are polished, T-tailed, and then the fragments are ligated to bisulfite-resistant sequence adapters. ( C ) The templates are then melted and subjected to partial bisulfite conversion (Materials and Methods). The conversion process randomly deaminates unmethylated cytosines (blue C), converting some proportion to uracil (red U). Each double-stranded molecule results in two templates, one from each strand. ( D ) We then sample the converted templates and PCR amplify to generate a sequencing library. During amplification uracil is copied as thymine (red T). ( E ) The library is then paired-end sequenced on an Illumina machine. Because of asymmetries in the sequencing primers, read one shows C to T conversion while read 2 shows G to A conversions. We then fully convert the reads ( F ) and map them to two fully converted genomes ( G ): one where every C in the reference is converted to a T and one where every G is converted to an A. Reads originating from a top strand map to the C to T reference with read 1 reading in the forward direction and read 2 in the reverse, while bottom strand reads map to the G to A reference with read 2 mapping forward and read 1 in reverse. ( H ) The reads are binned by genomic locus and strand. Mutable positions and their conversions are recorded as a bit pattern. We then use those patterns to cluster the reads (see Figure 3 ).
Figure Legend Snippet: muSeq applied to a genomic representation. We demonstrate the application of muSeq to a genomic representation by tracing two fragments (X and Y) from a single genomic locus through the molecular and informatics processing steps. The full experiment comprises 160,000 genomic loci and about 100 fragments per locus. ( A ) We first generate a representation from genomic DNA using PstI. ( B ) The ends of restriction fragments are polished, T-tailed, and then the fragments are ligated to bisulfite-resistant sequence adapters. ( C ) The templates are then melted and subjected to partial bisulfite conversion (Materials and Methods). The conversion process randomly deaminates unmethylated cytosines (blue C), converting some proportion to uracil (red U). Each double-stranded molecule results in two templates, one from each strand. ( D ) We then sample the converted templates and PCR amplify to generate a sequencing library. During amplification uracil is copied as thymine (red T). ( E ) The library is then paired-end sequenced on an Illumina machine. Because of asymmetries in the sequencing primers, read one shows C to T conversion while read 2 shows G to A conversions. We then fully convert the reads ( F ) and map them to two fully converted genomes ( G ): one where every C in the reference is converted to a T and one where every G is converted to an A. Reads originating from a top strand map to the C to T reference with read 1 reading in the forward direction and read 2 in the reverse, while bottom strand reads map to the G to A reference with read 2 mapping forward and read 1 in reverse. ( H ) The reads are binned by genomic locus and strand. Mutable positions and their conversions are recorded as a bit pattern. We then use those patterns to cluster the reads (see Figure 3 ).

Techniques Used: Sequencing, Polymerase Chain Reaction, Amplification

9) Product Images from "Selection and characterization of DNA aptamer against glucagon receptor by cell-SELEX"

Article Title: Selection and characterization of DNA aptamer against glucagon receptor by cell-SELEX

Journal: Scientific Reports

doi: 10.1038/s41598-017-05840-w

Scheme of cell-SELEX for GCGR-expressing CHO-K1 cells (CHO-GCGR). The ssDNA library was incubated with Mock cells as negative selection to remove the cell-binding ssDNA. The unbound ssDNA was incubated with GCGR-expressing CHO-K1 cells (CHO-GCGR) for positive selection. After washing, the bound DNA was eluted and amplified by PCR for next-round selection. The evolved ssDNA pool was sequenced to identify the aptamer candidates in the last round of selection.
Figure Legend Snippet: Scheme of cell-SELEX for GCGR-expressing CHO-K1 cells (CHO-GCGR). The ssDNA library was incubated with Mock cells as negative selection to remove the cell-binding ssDNA. The unbound ssDNA was incubated with GCGR-expressing CHO-K1 cells (CHO-GCGR) for positive selection. After washing, the bound DNA was eluted and amplified by PCR for next-round selection. The evolved ssDNA pool was sequenced to identify the aptamer candidates in the last round of selection.

Techniques Used: Expressing, Incubation, Selection, Binding Assay, Amplification, Polymerase Chain Reaction

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

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

Journal: Genome Research

doi: 10.1101/gr.196857.115

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

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

11) Product Images from "Reverse Engineering of Vaccine Antigens Using High Throughput Sequencing-enhanced mRNA Display"

Article Title: Reverse Engineering of Vaccine Antigens Using High Throughput Sequencing-enhanced mRNA Display

Journal: EBioMedicine

doi: 10.1016/j.ebiom.2015.06.021

mRNA display selection combined with HTS. The DNA library used contains a T7 promoter (T7), a CMV Translation enhancer (TE), a 15 or 27-mer random region ((Trimer) 15or27 ) and a constant region (3′ constant) encoding the peptide QLRNSCA. “Trimer” represents the mixture of 20 trimer (codon) phosphoramidites (Glen Research), each encoding one amino acid. In vitro transcription, ligation to a puromycin linker (green), in vitro translation and reverse transcription (RT) were performed as described in the Materials and methods section. mRNA/DNA–peptide fusions were applied to protein G magnetic beads (ProG beads) complexed with monoclonal antibodies (mAb) for selection. The regenerated DNA library was converted to an Illumina sequencing library by PCR amplification using forward and reverse primers containing Illumina adapters and subjected to MiSeq sequencing.
Figure Legend Snippet: mRNA display selection combined with HTS. The DNA library used contains a T7 promoter (T7), a CMV Translation enhancer (TE), a 15 or 27-mer random region ((Trimer) 15or27 ) and a constant region (3′ constant) encoding the peptide QLRNSCA. “Trimer” represents the mixture of 20 trimer (codon) phosphoramidites (Glen Research), each encoding one amino acid. In vitro transcription, ligation to a puromycin linker (green), in vitro translation and reverse transcription (RT) were performed as described in the Materials and methods section. mRNA/DNA–peptide fusions were applied to protein G magnetic beads (ProG beads) complexed with monoclonal antibodies (mAb) for selection. The regenerated DNA library was converted to an Illumina sequencing library by PCR amplification using forward and reverse primers containing Illumina adapters and subjected to MiSeq sequencing.

Techniques Used: Selection, In Vitro, Ligation, Magnetic Beads, Sequencing, Polymerase Chain Reaction, Amplification

12) Product Images from "Multiplexed in vivo homology-directed repair and tumor barcoding enables parallel quantification of Kras variant oncogenicity"

Article Title: Multiplexed in vivo homology-directed repair and tumor barcoding enables parallel quantification of Kras variant oncogenicity

Journal: Nature Communications

doi: 10.1038/s41467-017-01519-y

Design and validation of an AAV targeting vector library to introduce all Kras codon 12 and 13 single-nucleotide non-synonymous point mutations into somatic mouse cells in a multiplexed manner. a AAV vector pool for Cas9-mediated HDR into the endogenous Kras locus (AAV- Kras HDR /sg Kras / Cre ). Each vector contained an HDR template with 1 of 12 non-synonymous Kras mutations at codons 12 and 13 (or wild-type Kras ), silent mutations within the PAM (boxed sequence) and sgRNA homology region (PAM*), and a random 8-nucleotide barcode within the wobble positions of the adjacent codons for stable DNA barcoding of individual tumors. b Representation of each Kras HDR allele in the AAV- Kras HDR /sg Kras / Cre plasmid library. c Diversity of the barcode region in the AAV- Kras HDR /sg Kras / Cre plasmid library. d Schematic of the experiment to test for HDR bias. A Cas9-expressing cell line was transduced with AAV- Kras HDR /sg Kras / Cre and Kras HDR alleles were sequenced to quantify HDR events. e Schematic of the PCR strategy to specifically amplify Kras HDR alleles introduced into the genome via HDR. Forward primer 1 (F1) binds to the sequence containing the 3 PAM* mutations, while reverse primer 1 (R1) binds the endogenous Kras locus, outside the sequence present in the homology arm of the Kras HDR template. F2 binds to the Illumina adaptor added by F1, R2 binds to a region near exon 2, and R3 binds to the Illumina adapter added in the same reaction by R2. f Frequency of HDR events for each Kras HDR allele plotted against the initial frequency of each Kras mutant allele in the AAV- Kras HDR /sg Kras / Cre plasmid library. High correlation between the initial plasmid library and the representation of mutant Kras alleles following HDR suggests little to no HDR bias
Figure Legend Snippet: Design and validation of an AAV targeting vector library to introduce all Kras codon 12 and 13 single-nucleotide non-synonymous point mutations into somatic mouse cells in a multiplexed manner. a AAV vector pool for Cas9-mediated HDR into the endogenous Kras locus (AAV- Kras HDR /sg Kras / Cre ). Each vector contained an HDR template with 1 of 12 non-synonymous Kras mutations at codons 12 and 13 (or wild-type Kras ), silent mutations within the PAM (boxed sequence) and sgRNA homology region (PAM*), and a random 8-nucleotide barcode within the wobble positions of the adjacent codons for stable DNA barcoding of individual tumors. b Representation of each Kras HDR allele in the AAV- Kras HDR /sg Kras / Cre plasmid library. c Diversity of the barcode region in the AAV- Kras HDR /sg Kras / Cre plasmid library. d Schematic of the experiment to test for HDR bias. A Cas9-expressing cell line was transduced with AAV- Kras HDR /sg Kras / Cre and Kras HDR alleles were sequenced to quantify HDR events. e Schematic of the PCR strategy to specifically amplify Kras HDR alleles introduced into the genome via HDR. Forward primer 1 (F1) binds to the sequence containing the 3 PAM* mutations, while reverse primer 1 (R1) binds the endogenous Kras locus, outside the sequence present in the homology arm of the Kras HDR template. F2 binds to the Illumina adaptor added by F1, R2 binds to a region near exon 2, and R3 binds to the Illumina adapter added in the same reaction by R2. f Frequency of HDR events for each Kras HDR allele plotted against the initial frequency of each Kras mutant allele in the AAV- Kras HDR /sg Kras / Cre plasmid library. High correlation between the initial plasmid library and the representation of mutant Kras alleles following HDR suggests little to no HDR bias

Techniques Used: Plasmid Preparation, Introduce, Sequencing, Expressing, Transduction, Polymerase Chain Reaction, Mutagenesis

13) Product Images from "Influence of Heat Events on the Composition of Airborne Bacterial Communities in Urban Ecosystems"

Article Title: Influence of Heat Events on the Composition of Airborne Bacterial Communities in Urban Ecosystems

Journal: International Journal of Environmental Research and Public Health

doi: 10.3390/ijerph15102295

Airborne bacterial 16S rRNA gene copies as determined by quantitative real-time PCR beginning and end the heat event at four selected sampling sites in Hangzhou.
Figure Legend Snippet: Airborne bacterial 16S rRNA gene copies as determined by quantitative real-time PCR beginning and end the heat event at four selected sampling sites in Hangzhou.

Techniques Used: Real-time Polymerase Chain Reaction, Sampling

14) Product Images from "Analysis of Fish Commonly Sold in Local Supermarkets Reveals the Presence of Pathogenic and Multidrug-Resistant Bacterial Communities"

Article Title: Analysis of Fish Commonly Sold in Local Supermarkets Reveals the Presence of Pathogenic and Multidrug-Resistant Bacterial Communities

Journal: Microbiology Insights

doi: 10.1177/1178636118786925

The 20 most abundant genera out of a total of 168 genera detected are shown. PCR indicates polymerase chain reaction; rRNA, ribosomal RNA.
Figure Legend Snippet: The 20 most abundant genera out of a total of 168 genera detected are shown. PCR indicates polymerase chain reaction; rRNA, ribosomal RNA.

Techniques Used: Polymerase Chain Reaction

15) Product Images from "GSK3β is a key regulator of the ROS-dependent necrotic death induced by the quinone DMNQ"

Article Title: GSK3β is a key regulator of the ROS-dependent necrotic death induced by the quinone DMNQ

Journal: Cell Death & Disease

doi: 10.1038/s41419-019-2202-0

Screening protocol and targets validation. a Schematic representation of the screening protocol. 66 × 10 6 millions of cells were infected with the lentiviral particles containing the shRNA plasmids with a MOI of 0,1 for 2 days. For the selection of the expressing-shRNAs, cells were first grown in the presence of puromycin (2 μg/ml) for additional 3 days and subsequently treated with G5 (2.5 μM) for 60 h. Surviving cells were harvested to recover the genomic DNA. b Representation of the most enriched hits. Data are represented as fold enrichment respect to the median of all shRNAs. The enrichment is relative to the second most abundant shRNA. Only shRNA with a fold increase > 3 are shown. c Hits that are in relationship with GSK3β as up-stream regulators or downstream effectors. d U87MG cells were silenced for CAPN1 and PP2AC and, after 48 h from transfection, they were treated with G5 (2.5 μM) for 24 h. Cell death was calculated as percentage of cells positive to PI staining using cytofluorimetric analysis. Data are from three experiments; +SD. e Cellular lysates from the silenced cells were analyzed by immunoblot. Antibodies anti-CAPN1, anti-PP2AC, and anti-Actin (as loading control) were used as indicated. f U87MG and U87MG-BCLXL cells were silenced for GSK3β. After 48 h they were treated with G5 (2.5 μM) for further 24 h. Cell death was calculated as percentage of cells positive to PI staining using cytofluorimetric analysis. Data are from three experiments; +SD. g qRT-PCR analysis of GSK3β mRNA levels in silenced cells. Data were from three experiments; +SD. h Immunoblot analysis of GSK3β levels in the indicated clones of U87MG cells, selected after CRISPR/Cas9 mediated knock-out. Actin was used as loading control. i Cytofluorimetric analysis were performed to measure the % of PI positivity in the different U87MG clones treated with G5 (10 µM) for 24 h. Data are presented as mean ± SD. n = 3.
Figure Legend Snippet: Screening protocol and targets validation. a Schematic representation of the screening protocol. 66 × 10 6 millions of cells were infected with the lentiviral particles containing the shRNA plasmids with a MOI of 0,1 for 2 days. For the selection of the expressing-shRNAs, cells were first grown in the presence of puromycin (2 μg/ml) for additional 3 days and subsequently treated with G5 (2.5 μM) for 60 h. Surviving cells were harvested to recover the genomic DNA. b Representation of the most enriched hits. Data are represented as fold enrichment respect to the median of all shRNAs. The enrichment is relative to the second most abundant shRNA. Only shRNA with a fold increase > 3 are shown. c Hits that are in relationship with GSK3β as up-stream regulators or downstream effectors. d U87MG cells were silenced for CAPN1 and PP2AC and, after 48 h from transfection, they were treated with G5 (2.5 μM) for 24 h. Cell death was calculated as percentage of cells positive to PI staining using cytofluorimetric analysis. Data are from three experiments; +SD. e Cellular lysates from the silenced cells were analyzed by immunoblot. Antibodies anti-CAPN1, anti-PP2AC, and anti-Actin (as loading control) were used as indicated. f U87MG and U87MG-BCLXL cells were silenced for GSK3β. After 48 h they were treated with G5 (2.5 μM) for further 24 h. Cell death was calculated as percentage of cells positive to PI staining using cytofluorimetric analysis. Data are from three experiments; +SD. g qRT-PCR analysis of GSK3β mRNA levels in silenced cells. Data were from three experiments; +SD. h Immunoblot analysis of GSK3β levels in the indicated clones of U87MG cells, selected after CRISPR/Cas9 mediated knock-out. Actin was used as loading control. i Cytofluorimetric analysis were performed to measure the % of PI positivity in the different U87MG clones treated with G5 (10 µM) for 24 h. Data are presented as mean ± SD. n = 3.

Techniques Used: Infection, shRNA, Selection, Expressing, Transfection, Staining, Quantitative RT-PCR, Clone Assay, CRISPR, Knock-Out

16) Product Images from "Genome editing in primary cells and in vivo using viral-derived Nanoblades loaded with Cas9-sgRNA ribonucleoproteins"

Article Title: Genome editing in primary cells and in vivo using viral-derived Nanoblades loaded with Cas9-sgRNA ribonucleoproteins

Journal: Nature Communications

doi: 10.1038/s41467-018-07845-z

Genome editing in primary cells transduced with Nanoblades. a Left panel, editing efficiency at the EMX1 locus (measured by high-throughput sequencing on the Illumina Miseq platform) of human-induced pluripotent stem cells (hiPSCs) transduced with Nanoblades targeting human EMX1 ( n = 3). Right panel, expression of pluripotency markers measured by qPCR in control cells and cells transduced with Nanoblades targeting EMX1 ( n = 3). b Left and middle panels, fluorescence microscopy and FACS analysis of GFP expressing BMDMs transduced at the bone marrow stage (day 0 after bone marrow collection) with control Nanoblades or Nanoblades targeting the GFP- coding sequence ( n = 3). Right top panel, T7 endonuclease assay against the GFP sequence from Nanoblades-treated BMDMs. Right bottom panel, cytokine expression levels (measured by qPCR) in untreated or Nanoblade-treated cells upon LPS stimulation ( n = 4). c T7 endonuclease assay against mouse Fto or human EMX1 analysis of the Sanger sequencing electropherograms for each PCR amplicon d Left panel, excision of a 160 bp DNA fragment of MYD88 using Nanoblades. Middle panel PCR results obtained in human primary hepatocytes transduced with Nanoblades. Right-panel (top), FACS analysis of CD34+ cells purified from human cord-blood. Bottom, genome editing at the MYD88 locus assessed by PCR in untreated and Nanoblades-treated CD34+ cells. Error bars in all figures correspond to standard deviation
Figure Legend Snippet: Genome editing in primary cells transduced with Nanoblades. a Left panel, editing efficiency at the EMX1 locus (measured by high-throughput sequencing on the Illumina Miseq platform) of human-induced pluripotent stem cells (hiPSCs) transduced with Nanoblades targeting human EMX1 ( n = 3). Right panel, expression of pluripotency markers measured by qPCR in control cells and cells transduced with Nanoblades targeting EMX1 ( n = 3). b Left and middle panels, fluorescence microscopy and FACS analysis of GFP expressing BMDMs transduced at the bone marrow stage (day 0 after bone marrow collection) with control Nanoblades or Nanoblades targeting the GFP- coding sequence ( n = 3). Right top panel, T7 endonuclease assay against the GFP sequence from Nanoblades-treated BMDMs. Right bottom panel, cytokine expression levels (measured by qPCR) in untreated or Nanoblade-treated cells upon LPS stimulation ( n = 4). c T7 endonuclease assay against mouse Fto or human EMX1 analysis of the Sanger sequencing electropherograms for each PCR amplicon d Left panel, excision of a 160 bp DNA fragment of MYD88 using Nanoblades. Middle panel PCR results obtained in human primary hepatocytes transduced with Nanoblades. Right-panel (top), FACS analysis of CD34+ cells purified from human cord-blood. Bottom, genome editing at the MYD88 locus assessed by PCR in untreated and Nanoblades-treated CD34+ cells. Error bars in all figures correspond to standard deviation

Techniques Used: Transduction, Next-Generation Sequencing, Expressing, Real-time Polymerase Chain Reaction, Fluorescence, Microscopy, FACS, Sequencing, Polymerase Chain Reaction, Amplification, Purification, Standard Deviation

17) Product Images from "iCLIP reveals the function of hnRNP particles in splicing at individual nucleotide resolution"

Article Title: iCLIP reveals the function of hnRNP particles in splicing at individual nucleotide resolution

Journal: Nature structural & molecular biology

doi: 10.1038/nsmb.1838

iCLIP identifies hnRNP C cross-link nucleotides on RNAs. (a) Schematic representation of the iCLIP protocol. After UV irradiation, the covalently linked RNA is co-immunoprecipitated with the RNA-binding protein (RBP) and ligated to an RNA adapter at the 3′ end. Proteinase K digestion leaves a covalently bound polypeptide fragment on the RNA that causes premature truncation of reverse transcription (RT) at the cross-link site. The red bar indicates the last nucleotide added during reverse transcription. Resulting cDNA molecules are circularized, linearized, PCR-amplified and subjected to high-throughput sequencing. The first nucleotides of each sequence contain the barcode followed by the nucleotide where cDNAs truncated during reverse transcription. (b) Reproducibility of cross-link nucleotide positions. Percentage of cross-link nucleotides with a given cDNA count that were identified in at least two (circles) or all three experiments (triangles) are shown. The percentage of reproduced cross-link nucleotides increased with the incidence of hnRNP C cross-linking (cDNA count). (c) Reproducibility of sequence composition at cross-link nucleotides. Frequencies of pentanucleotides overlapping with cross-link nucleotides are shown for the three replicate experiments ( R 2 = 0.9996, R 2 = 0.9987 and R 2 = 0.9996) with the sequence shown for the four most highly enriched pentanucleotides. 42% of cross-link nucleotides overlap with UUUUU in all three replicate experiments.
Figure Legend Snippet: iCLIP identifies hnRNP C cross-link nucleotides on RNAs. (a) Schematic representation of the iCLIP protocol. After UV irradiation, the covalently linked RNA is co-immunoprecipitated with the RNA-binding protein (RBP) and ligated to an RNA adapter at the 3′ end. Proteinase K digestion leaves a covalently bound polypeptide fragment on the RNA that causes premature truncation of reverse transcription (RT) at the cross-link site. The red bar indicates the last nucleotide added during reverse transcription. Resulting cDNA molecules are circularized, linearized, PCR-amplified and subjected to high-throughput sequencing. The first nucleotides of each sequence contain the barcode followed by the nucleotide where cDNAs truncated during reverse transcription. (b) Reproducibility of cross-link nucleotide positions. Percentage of cross-link nucleotides with a given cDNA count that were identified in at least two (circles) or all three experiments (triangles) are shown. The percentage of reproduced cross-link nucleotides increased with the incidence of hnRNP C cross-linking (cDNA count). (c) Reproducibility of sequence composition at cross-link nucleotides. Frequencies of pentanucleotides overlapping with cross-link nucleotides are shown for the three replicate experiments ( R 2 = 0.9996, R 2 = 0.9987 and R 2 = 0.9996) with the sequence shown for the four most highly enriched pentanucleotides. 42% of cross-link nucleotides overlap with UUUUU in all three replicate experiments.

Techniques Used: Irradiation, Immunoprecipitation, RNA Binding Assay, Polymerase Chain Reaction, Amplification, Next-Generation Sequencing, Sequencing

18) Product Images from "iCLIP identifies novel roles for SAFB1 in regulating RNA processing and neuronal function"

Article Title: iCLIP identifies novel roles for SAFB1 in regulating RNA processing and neuronal function

Journal: BMC Biology

doi: 10.1186/s12915-015-0220-7

Exon array analyses following SAFB1 knockdown. Samples were prepared from SYSY5Y cells following siRNA-mediated SAFB1 knockdown and 1.0 ST Affymetrix high-resolution exon arrays used to analyse effects on splicing. AltAnalyse was used to interrogate the microarray data for alternative splicing events and the significant changes are shown ( a ). RT-PCR was used to confirm the splicing events identified by arrays ( b and c ). Primer pairs were generated to distinguish isoforms and three independent experiments were carried out in triplicate. SAFB1 knockdown mediated statistically significant changes in expression of the gene transcripts shown in (b and c). Statistical analysis was by ANOVA followed by post-hoc t -tests. ** P
Figure Legend Snippet: Exon array analyses following SAFB1 knockdown. Samples were prepared from SYSY5Y cells following siRNA-mediated SAFB1 knockdown and 1.0 ST Affymetrix high-resolution exon arrays used to analyse effects on splicing. AltAnalyse was used to interrogate the microarray data for alternative splicing events and the significant changes are shown ( a ). RT-PCR was used to confirm the splicing events identified by arrays ( b and c ). Primer pairs were generated to distinguish isoforms and three independent experiments were carried out in triplicate. SAFB1 knockdown mediated statistically significant changes in expression of the gene transcripts shown in (b and c). Statistical analysis was by ANOVA followed by post-hoc t -tests. ** P

Techniques Used: Microarray, Reverse Transcription Polymerase Chain Reaction, Generated, Expressing

SAFB1 knockdown mediates changes in the expression of specific RNA isoforms. RT-PCR analysis of genes with crosslinks identified by iCLIP was performed in such a way as to examine the possibility of transcript-specific alterations. a RNA was isolated from SHSY5Y cells following siRNA mediated SAFB1 knockdown and SAFB1 and SAFB2 RNA levels measured. b Specific primer sets were designed and used to measure the expression of NCAM1, SYNPO2, ASTN2 and PDE4B isoforms. The results were obtained following three independent experiments. Statistical analysis was by ANOVA followed by post-hoc t -tests. * P
Figure Legend Snippet: SAFB1 knockdown mediates changes in the expression of specific RNA isoforms. RT-PCR analysis of genes with crosslinks identified by iCLIP was performed in such a way as to examine the possibility of transcript-specific alterations. a RNA was isolated from SHSY5Y cells following siRNA mediated SAFB1 knockdown and SAFB1 and SAFB2 RNA levels measured. b Specific primer sets were designed and used to measure the expression of NCAM1, SYNPO2, ASTN2 and PDE4B isoforms. The results were obtained following three independent experiments. Statistical analysis was by ANOVA followed by post-hoc t -tests. * P

Techniques Used: Expressing, Reverse Transcription Polymerase Chain Reaction, Isolation

SAFB1 regulates splicing from wild type and mutant NCAM1 minigenes. a The distribution of SAFB1 iCLIP tags within the NCAM1 minigene is shown and an arrow indicates the position of exon 9 in transcript variant 5. b The schematic indicates the composition of the minigenes and splice forms generated. The sequence of wild type exon 9, the location of the GAA/AAG and AGA motifs, and the mutations introduced to alter all AAG/AGA/GAA are shown. c PCR analyses using exon-specific spanning primers was used to analyse the effect of SAFB1 knockdown on the expression of the NCAM1 isoforms (NCAM1 7–10, 8–10 and 9–10) in the wild type and mutant minigenes. The open bars represent expression of the transcript indicated with non-targeting control knockdown and the closed bars following SAFB1 knockdown. Values are means ± SEM, statistical analysis was performed by ANOVA and post-hoc t -tests * P
Figure Legend Snippet: SAFB1 regulates splicing from wild type and mutant NCAM1 minigenes. a The distribution of SAFB1 iCLIP tags within the NCAM1 minigene is shown and an arrow indicates the position of exon 9 in transcript variant 5. b The schematic indicates the composition of the minigenes and splice forms generated. The sequence of wild type exon 9, the location of the GAA/AAG and AGA motifs, and the mutations introduced to alter all AAG/AGA/GAA are shown. c PCR analyses using exon-specific spanning primers was used to analyse the effect of SAFB1 knockdown on the expression of the NCAM1 isoforms (NCAM1 7–10, 8–10 and 9–10) in the wild type and mutant minigenes. The open bars represent expression of the transcript indicated with non-targeting control knockdown and the closed bars following SAFB1 knockdown. Values are means ± SEM, statistical analysis was performed by ANOVA and post-hoc t -tests * P

Techniques Used: Mutagenesis, Variant Assay, Generated, Sequencing, Polymerase Chain Reaction, Expressing

19) Product Images from "Gibberellin Promotes Bolting and Flowering via the Floral Integrators RsFT and RsSOC1-1 under Marginal Vernalization in Radish"

Article Title: Gibberellin Promotes Bolting and Flowering via the Floral Integrators RsFT and RsSOC1-1 under Marginal Vernalization in Radish

Journal: Plants

doi: 10.3390/plants9050594

Quantitative PCR analysis of flowering time DEGs in the two inbred radish lines in response to gibberellin. Total RNA was isolated from shoots of NH-JS1 and NH-JS2 inbred lines 6 days after treatment with or without GA (0 or 10 mM) spray application. The complementary DNA was synthesized from total RNA. Upregulated and downregulated genes are grouped according to biological pathways determined from gene ontology analysis. Flowering time genes involved in GA pathway ( A ), photoperiod pathway ( B ), vernalization pathway ( C ) and flowering integrators ( D ). The qPCR values were normalized relative to RsACT1 (actin) expression level. Error bars represent ± standard error of biological triplicates. Asterisks indicate statistically significant differences (NH-JS2 vs. NH-JS1 and NH-JS2_GA vs. NH-JS1_GA; Student’s t test; * p
Figure Legend Snippet: Quantitative PCR analysis of flowering time DEGs in the two inbred radish lines in response to gibberellin. Total RNA was isolated from shoots of NH-JS1 and NH-JS2 inbred lines 6 days after treatment with or without GA (0 or 10 mM) spray application. The complementary DNA was synthesized from total RNA. Upregulated and downregulated genes are grouped according to biological pathways determined from gene ontology analysis. Flowering time genes involved in GA pathway ( A ), photoperiod pathway ( B ), vernalization pathway ( C ) and flowering integrators ( D ). The qPCR values were normalized relative to RsACT1 (actin) expression level. Error bars represent ± standard error of biological triplicates. Asterisks indicate statistically significant differences (NH-JS2 vs. NH-JS1 and NH-JS2_GA vs. NH-JS1_GA; Student’s t test; * p

Techniques Used: Real-time Polymerase Chain Reaction, Isolation, Synthesized, Expressing

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

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

Journal: Molecular Systems Biology

doi: 10.15252/msb.20145776

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

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

21) Product Images from "Stress Granules Contain Rbfox2 with Cell Cycle-related mRNAs"

Article Title: Stress Granules Contain Rbfox2 with Cell Cycle-related mRNAs

Journal: Scientific Reports

doi: 10.1038/s41598-017-11651-w

Identification of Rbfox2 target RNAs using RIP-seq analysis. ( a ) Correlation of RNA levels (log 2 ( FPKM RIP - seq )) between two replicates. Venn diagram indicates the total number of Rbfox2 binding RNAs by both experimental conditions (RIP-seq-A and RIP-seq-B). ( b ) The distribution of Rbfox2 binding sites. ( c ) Correlation of expression level log fold change between Rbfox2 RIP-seq and control RNA-sequencing. The dashed rectangle indicates enriched RNAs (fold change ≥ 2) in Rbfox2 RIP-seq compared to control. ( d ) Gene Ontology (GO) analysis of enriched RNAs shown in the dashed rectangle of ( c ). The 14 most enriched GO biological process categories are shown. ( e ) An integrative genomics viewer (IGV) browser snapshot of Rbfox2 occupancy within the RB1 locus in two biological replicates (RIP-seq-A and RIP-seq-B) for RIP-seq analysis. ( f ) Phylogenetic conservation of 3′ UTR of RB1. Conservation of the RB1 targeting 3′ UTR is shown by the phastCons track on the UCSC Genome Browser in the human hg19 genome. The phastCons score estimates the probability of each nucleotide, using a hidden markov model-based method, belonging to conserved elements. This track shows multiple alignments of 100 vertebrate species and measurements of evolution conservation. ( g ) After treatment of HeLa cells with sodium arsenite, RIP-qRT-PCR (IP followed by qRT-PCR) analysis was used to measure the levels of enrichment of RB1 mRNA and GAPDH mRNA associated with immune-complexes using anti-Rbfox2 (αRbfox2) or control IgG; the data represented as enrichment of each mRNA in immuno-complexes were compared with 5% input. Mean values ± SD (error bars) are shown for three independent experiments. * P
Figure Legend Snippet: Identification of Rbfox2 target RNAs using RIP-seq analysis. ( a ) Correlation of RNA levels (log 2 ( FPKM RIP - seq )) between two replicates. Venn diagram indicates the total number of Rbfox2 binding RNAs by both experimental conditions (RIP-seq-A and RIP-seq-B). ( b ) The distribution of Rbfox2 binding sites. ( c ) Correlation of expression level log fold change between Rbfox2 RIP-seq and control RNA-sequencing. The dashed rectangle indicates enriched RNAs (fold change ≥ 2) in Rbfox2 RIP-seq compared to control. ( d ) Gene Ontology (GO) analysis of enriched RNAs shown in the dashed rectangle of ( c ). The 14 most enriched GO biological process categories are shown. ( e ) An integrative genomics viewer (IGV) browser snapshot of Rbfox2 occupancy within the RB1 locus in two biological replicates (RIP-seq-A and RIP-seq-B) for RIP-seq analysis. ( f ) Phylogenetic conservation of 3′ UTR of RB1. Conservation of the RB1 targeting 3′ UTR is shown by the phastCons track on the UCSC Genome Browser in the human hg19 genome. The phastCons score estimates the probability of each nucleotide, using a hidden markov model-based method, belonging to conserved elements. This track shows multiple alignments of 100 vertebrate species and measurements of evolution conservation. ( g ) After treatment of HeLa cells with sodium arsenite, RIP-qRT-PCR (IP followed by qRT-PCR) analysis was used to measure the levels of enrichment of RB1 mRNA and GAPDH mRNA associated with immune-complexes using anti-Rbfox2 (αRbfox2) or control IgG; the data represented as enrichment of each mRNA in immuno-complexes were compared with 5% input. Mean values ± SD (error bars) are shown for three independent experiments. * P

Techniques Used: Binding Assay, Expressing, RNA Sequencing Assay, Quantitative RT-PCR

Rbfox2 affects RB1 protein expression under cellular stress. ( a ) HeLa cells were transfected with siCon. or siRbfox2 for 36 hr. Representative immunoblot showing the effects of siRNA transfections on protein levels of Rbfox2 (green) and Gapdh (red). ( b ) The steady- state levels of RB1 and GAPDH mRNAs were measured by qRT-PCR using total RNA from control HeLa cells (siCon.) and Rbfox2-deficient HeLa cells (siRbfox2) treated for 40 min with or without sodium arsenite (500 μM), followed by treatment for 0–3 hr with actinomycin D (2.5 μg/ml) in arsenite-free media. * P
Figure Legend Snippet: Rbfox2 affects RB1 protein expression under cellular stress. ( a ) HeLa cells were transfected with siCon. or siRbfox2 for 36 hr. Representative immunoblot showing the effects of siRNA transfections on protein levels of Rbfox2 (green) and Gapdh (red). ( b ) The steady- state levels of RB1 and GAPDH mRNAs were measured by qRT-PCR using total RNA from control HeLa cells (siCon.) and Rbfox2-deficient HeLa cells (siRbfox2) treated for 40 min with or without sodium arsenite (500 μM), followed by treatment for 0–3 hr with actinomycin D (2.5 μg/ml) in arsenite-free media. * P

Techniques Used: Expressing, Transfection, Quantitative RT-PCR

22) Product Images from "Reagent and laboratory contamination can critically impact sequence-based microbiome analyses"

Article Title: Reagent and laboratory contamination can critically impact sequence-based microbiome analyses

Journal: BMC Biology

doi: 10.1186/s12915-014-0087-z

Summary of 16S rRNA gene sequencing taxonomic assignment from ten-fold diluted pure cultures and controls. Undiluted DNA extractions contained approximately 10 8 cells, and controls (annotated in the Figure with 'con') were template-free PCRs. DNA was extracted at ICL, UB and WTSI laboratories and amplified with 40 PCR cycles. Each column represents a single sample; sections (a) and (b) describe the same samples at different taxonomic levels. a) Proportion of S. bongori sequence reads in black. The proportional abundance of non- Salmonella reads at the Class level is indicated by other colours. As the sample becomes more dilute, the proportion of the sequenced bacterial amplicons from the cultured microorganism decreases and contaminants become more dominant. b) Abundance of genera which make up > 0.5% of the results from at least one laboratory, excluding S. bongori . The profiles of the non- Salmonella reads within each laboratory/kit batch are consistent but differ between sites.
Figure Legend Snippet: Summary of 16S rRNA gene sequencing taxonomic assignment from ten-fold diluted pure cultures and controls. Undiluted DNA extractions contained approximately 10 8 cells, and controls (annotated in the Figure with 'con') were template-free PCRs. DNA was extracted at ICL, UB and WTSI laboratories and amplified with 40 PCR cycles. Each column represents a single sample; sections (a) and (b) describe the same samples at different taxonomic levels. a) Proportion of S. bongori sequence reads in black. The proportional abundance of non- Salmonella reads at the Class level is indicated by other colours. As the sample becomes more dilute, the proportion of the sequenced bacterial amplicons from the cultured microorganism decreases and contaminants become more dominant. b) Abundance of genera which make up > 0.5% of the results from at least one laboratory, excluding S. bongori . The profiles of the non- Salmonella reads within each laboratory/kit batch are consistent but differ between sites.

Techniques Used: Sequencing, Amplification, Polymerase Chain Reaction, Cell Culture

23) Product Images from "Genetic Screens Reveal FEN1 and APEX2 as BRCA2 Synthetic Lethal Targets"

Article Title: Genetic Screens Reveal FEN1 and APEX2 as BRCA2 Synthetic Lethal Targets

Journal: Molecular cell

doi: 10.1016/j.molcel.2018.12.008

FEN1 participates in MMEJ. (A) Schematic of MMEJ reporter. (B-D) Negative control siRNA-transfected cells from (E) were infected with lentivirus expressing I-SceI, grown for 5 d, and were FACS-sorted for E2-Crimson + GFP + populations (C) or GFP + populations (D). Genomic DNA was isolated from these FACS-sorted cell populations as well as total unsorted cells (B), and the E2-Crimson construct was PCR-amplified and adapted for Illumina sequencing. The results of 500K reads were plotted for each cell population as a percent of MMEJ or NHEJ, with the indicated size (bp) of microhomology utilized for repair. Restoration of the wild-type sequence is indicated with an asterisk. (E-F) U2-OS cells containing the MMEJ reporter were transfected with the indicated siRNAs and transduced with lentiviral I-SceI. The percent of E2-Crimson + GFP + cells was quantified by FACS after 5 d of growth. Error bars reflect variability of technical triplicates, and the number of asterisks indicates the statistical significance for the corresponding siRNA versus negative control siRNA (*=p
Figure Legend Snippet: FEN1 participates in MMEJ. (A) Schematic of MMEJ reporter. (B-D) Negative control siRNA-transfected cells from (E) were infected with lentivirus expressing I-SceI, grown for 5 d, and were FACS-sorted for E2-Crimson + GFP + populations (C) or GFP + populations (D). Genomic DNA was isolated from these FACS-sorted cell populations as well as total unsorted cells (B), and the E2-Crimson construct was PCR-amplified and adapted for Illumina sequencing. The results of 500K reads were plotted for each cell population as a percent of MMEJ or NHEJ, with the indicated size (bp) of microhomology utilized for repair. Restoration of the wild-type sequence is indicated with an asterisk. (E-F) U2-OS cells containing the MMEJ reporter were transfected with the indicated siRNAs and transduced with lentiviral I-SceI. The percent of E2-Crimson + GFP + cells was quantified by FACS after 5 d of growth. Error bars reflect variability of technical triplicates, and the number of asterisks indicates the statistical significance for the corresponding siRNA versus negative control siRNA (*=p

Techniques Used: Negative Control, Transfection, Infection, Expressing, FACS, Isolation, Construct, Polymerase Chain Reaction, Amplification, Sequencing, Non-Homologous End Joining, Transduction

24) Product Images from "Ribosome maturation by the endoribonuclease YbeY stabilizes a type 3 secretion system transcript required for virulence of enterohemorrhagic Escherichia coli"

Article Title: Ribosome maturation by the endoribonuclease YbeY stabilizes a type 3 secretion system transcript required for virulence of enterohemorrhagic Escherichia coli

Journal: The Journal of Biological Chemistry

doi: 10.1074/jbc.RA117.000300

Translation stabilizes the espD transcript. A , Northern blot analysis of espD ( left panel ) and recA ( right panel ) in WT ZAP193, Δ ybeY , and the ybeY -HTF strain backgrounds. B , transcription and translation of the first gene in the LEE4 polycistronic operon, sepL , was measured using GFP fusions. Left , the transcriptional sepL -GFP fusion (pCDR8) containing the native promoter and 8 nt of the sepL 5′-UTR was measured in ZAP193 and Δ ybeY backgrounds under T3S-permissive conditions. Right , the translational sepL -GFP fusion (pDW6) containing the native promoter and entire sepL CDS fused to GFP was similarly measured under T3S-permissive conditions. Error bars represent S.E. from biological triplicates. AU , arbitrary units. C , Western blotting for the T3S needle tip protein (EspD) in whole cells grown with and without a subinhibitory concentration of the translation initiation inhibitor kasugamycin (50 μg·ml −1 ). RecA was used as a loading control ( bottom panel ). D , RNA stability assay of espD and recA mRNAs with and without translating ribosomes. Transcription was blocked in EHEC str. ZAP193 by adding 1 mg·ml −1 rifampicin ( Rif ) ( closed circles ) or rifampicin and 1 mg·ml −1 kasugamycin ( Kas ) ( open circles ) at time 0. The relative abundance of mRNAs was monitored using quantitative RT-PCR. Error bars represent S.E. from biological triplicates. E , binding profiles for RNase E ( dark blue ), Hfq ( blue ), YbeY ( dark green ), and controls ( green ) are shown for transcripts from the LEE (ORFs are in gray ; espD is highlighted in red ). Protein binding to positive ( top ) and negative strands ( bottom ) of the transcriptome are shown. The polycistronic LEE1–5 transcripts are indicated above and below relevant ORFs.
Figure Legend Snippet: Translation stabilizes the espD transcript. A , Northern blot analysis of espD ( left panel ) and recA ( right panel ) in WT ZAP193, Δ ybeY , and the ybeY -HTF strain backgrounds. B , transcription and translation of the first gene in the LEE4 polycistronic operon, sepL , was measured using GFP fusions. Left , the transcriptional sepL -GFP fusion (pCDR8) containing the native promoter and 8 nt of the sepL 5′-UTR was measured in ZAP193 and Δ ybeY backgrounds under T3S-permissive conditions. Right , the translational sepL -GFP fusion (pDW6) containing the native promoter and entire sepL CDS fused to GFP was similarly measured under T3S-permissive conditions. Error bars represent S.E. from biological triplicates. AU , arbitrary units. C , Western blotting for the T3S needle tip protein (EspD) in whole cells grown with and without a subinhibitory concentration of the translation initiation inhibitor kasugamycin (50 μg·ml −1 ). RecA was used as a loading control ( bottom panel ). D , RNA stability assay of espD and recA mRNAs with and without translating ribosomes. Transcription was blocked in EHEC str. ZAP193 by adding 1 mg·ml −1 rifampicin ( Rif ) ( closed circles ) or rifampicin and 1 mg·ml −1 kasugamycin ( Kas ) ( open circles ) at time 0. The relative abundance of mRNAs was monitored using quantitative RT-PCR. Error bars represent S.E. from biological triplicates. E , binding profiles for RNase E ( dark blue ), Hfq ( blue ), YbeY ( dark green ), and controls ( green ) are shown for transcripts from the LEE (ORFs are in gray ; espD is highlighted in red ). Protein binding to positive ( top ) and negative strands ( bottom ) of the transcriptome are shown. The polycistronic LEE1–5 transcripts are indicated above and below relevant ORFs.

Techniques Used: Northern Blot, Western Blot, Concentration Assay, Stability Assay, Quantitative RT-PCR, Binding Assay, Protein Binding

25) Product Images from "RNA structure inference through chemical mapping after accidental or intentional mutations"

Article Title: RNA structure inference through chemical mapping after accidental or intentional mutations

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

doi: 10.1073/pnas.1619897114

Workflow for M2-seq, mutate-and-map read-out by next-generation sequencing. DNA template is generated by PCR assembly of oligonucleotides, PCR from a plasmid, or error-prone PCR, potentially introducing deletions (white) or mutations (gold). Then, RNA is transcribed (potentially introducing further mutations, cyan), folded, and DMS-modified. Reverse transcription is performed under conditions that favor mutational read-through of DMS-modified nucleotides, recording those positions as mutations (magenta), and cDNAs are PCR-amplified to generate double-stranded DNA library. Libraries are subjected to next-generation sequencing, and resulting reads are analyzed by demultiplexing, alignment to reference sequences, and correlated mutation counting to generate an M2-seq dataset (simulated here). Double-stranded RNA helices give rise to cross-diagonal features in these maps that can be automatically recognized by M2-net and confirmed visually.
Figure Legend Snippet: Workflow for M2-seq, mutate-and-map read-out by next-generation sequencing. DNA template is generated by PCR assembly of oligonucleotides, PCR from a plasmid, or error-prone PCR, potentially introducing deletions (white) or mutations (gold). Then, RNA is transcribed (potentially introducing further mutations, cyan), folded, and DMS-modified. Reverse transcription is performed under conditions that favor mutational read-through of DMS-modified nucleotides, recording those positions as mutations (magenta), and cDNAs are PCR-amplified to generate double-stranded DNA library. Libraries are subjected to next-generation sequencing, and resulting reads are analyzed by demultiplexing, alignment to reference sequences, and correlated mutation counting to generate an M2-seq dataset (simulated here). Double-stranded RNA helices give rise to cross-diagonal features in these maps that can be automatically recognized by M2-net and confirmed visually.

Techniques Used: Next-Generation Sequencing, Generated, Polymerase Chain Reaction, Plasmid Preparation, Modification, Amplification, Mutagenesis

26) Product Images from "Analyzing and minimizing PCR amplification bias in Illumina sequencing libraries"

Article Title: Analyzing and minimizing PCR amplification bias in Illumina sequencing libraries

Journal: Genome Biology

doi: 10.1186/gb-2011-12-2-r18

'PER' genome-wide base composition bias curves . (a,b) Shown is the GC bias in Illumina reads from a 400-bp fragment library amplified using the standard PCR protocol (Phusion HF, short denaturation) on a fast-ramping thermocycler (red squares), Phusion HF with long denaturation and 2M betaine (black triangles), AccuPrime Taq HiFi with long denaturation and primer extension at 65°C (blue diamonds) or 60°C (purple diamonds). To calculate the observed to expected (unbiased) read coverage, the number of reads aligning to 50-bp windows at a given %GC was divided by the number of 50-bp windows that fall in this %GC category. This value was then normalized relative to the average value from 48% through 52% GC and plotted on a log 10 scale (a) or linear scale (b).
Figure Legend Snippet: 'PER' genome-wide base composition bias curves . (a,b) Shown is the GC bias in Illumina reads from a 400-bp fragment library amplified using the standard PCR protocol (Phusion HF, short denaturation) on a fast-ramping thermocycler (red squares), Phusion HF with long denaturation and 2M betaine (black triangles), AccuPrime Taq HiFi with long denaturation and primer extension at 65°C (blue diamonds) or 60°C (purple diamonds). To calculate the observed to expected (unbiased) read coverage, the number of reads aligning to 50-bp windows at a given %GC was divided by the number of 50-bp windows that fall in this %GC category. This value was then normalized relative to the average value from 48% through 52% GC and plotted on a log 10 scale (a) or linear scale (b).

Techniques Used: Genome Wide, Amplification, Polymerase Chain Reaction

Sequencing bias with PCR-amplified and PCR-free libraries . (a,b) Shown is the mean normalized coverage of 50-bp windows in the human genome having the GC-content indicated on the x-axis for a PCR-free (orange dots) and a PCR-amplified (blue diamonds) Illumina sequencing library. Both fragment libraries had approximately 180-bp inserts. The PCR amplification was performed with AccuPrime Taq HiFi (long denat., primer extension at 65°C). The coverage was plotted on a log 10 (a) and a linear scale (b). The data points at extremely high GC, where the reads from the PCR-free library had a mean base quality of less than Q20 (open symbols), were omitted in the middle panel (b). (c) The ratios of the two curves in (a,b), that is, the fold-increase in mean coverage by sequencing a PCR-free library instead of a PCR-amplified library. The shaded histogram is the %GC distribution of 50-bp windows in the human genome. More than 99.9% of all 50-bp windows in the genome contain 8% to 88% GC and received a less than 1.25-fold increase in coverage. Less than 0.01% of all 50-bp windows contain 90% or more GC. The open circles at 96% and 98% GC denote data for which the mean base quality of the reads from the PCR-free library was below Q20.
Figure Legend Snippet: Sequencing bias with PCR-amplified and PCR-free libraries . (a,b) Shown is the mean normalized coverage of 50-bp windows in the human genome having the GC-content indicated on the x-axis for a PCR-free (orange dots) and a PCR-amplified (blue diamonds) Illumina sequencing library. Both fragment libraries had approximately 180-bp inserts. The PCR amplification was performed with AccuPrime Taq HiFi (long denat., primer extension at 65°C). The coverage was plotted on a log 10 (a) and a linear scale (b). The data points at extremely high GC, where the reads from the PCR-free library had a mean base quality of less than Q20 (open symbols), were omitted in the middle panel (b). (c) The ratios of the two curves in (a,b), that is, the fold-increase in mean coverage by sequencing a PCR-free library instead of a PCR-amplified library. The shaded histogram is the %GC distribution of 50-bp windows in the human genome. More than 99.9% of all 50-bp windows in the genome contain 8% to 88% GC and received a less than 1.25-fold increase in coverage. Less than 0.01% of all 50-bp windows contain 90% or more GC. The open circles at 96% and 98% GC denote data for which the mean base quality of the reads from the PCR-free library was below Q20.

Techniques Used: Sequencing, Polymerase Chain Reaction, Amplification

Related Articles

Amplification:

Article Title: Influence of Heat Events on the Composition of Airborne Bacterial Communities in Urban Ecosystems
Article Snippet: .. PCR Amplification and Illumina Miseq PE300 Sequencing The V3–V4 regions of the 16S rRNA gene were PCR-amplified for Illumina Miseq PE300 sequencing. .. Amplicon PCR was performed in 25 μL volume reactions with 12.5 μL of 2× KAPA HiFi HotStart ReadyMix, 2.5 μL of microbial DNA, and 5 μL of each primer (Klindworth et al., 2013) using primers 341 F (CCTACGGGNGGCWGCAG) and 805 R (GACTACHVGGGTATCTAATCC).

Sample Prep:

Article Title: Next-generation DNA sequencing-based assay for measuring allelic expression imbalance (AEI) of candidate neuropsychiatric disorder genes in human brain
Article Snippet: .. Final steps in DNA sample preparation To prepare the DNA fragments for sequencing, the ligated gDNA- and cDNA-derived DNAs were independently PCR-amplified using Illumina Primers 1.1 and 1.2 and Phusion DNA polymerase (Finnzymes Oy). .. The PCR products were resolved by electrophoresis on 15% polyacrylamide gels and DNAs of the appropriate size extracted using QIAquick Gel Extraction Kits (Qiagen).

Synthesized:

Article Title: Ribosome maturation by the endoribonuclease YbeY stabilizes a type 3 secretion system transcript required for virulence of enterohemorrhagic Escherichia coli
Article Snippet: .. Linkers were ligated during purification, and cDNA libraries were synthesized from eluted RNA, PCR-amplified, and sequenced using the Illumina MiSeq platform. .. The YbeY binding profile was analyzed using the pyCRAC package , and statistically significant peaks were identified using pyCalculateFDR as described previously ( ) ( A ).

Next-Generation Sequencing:

Article Title: Detection of bacterial DNA from central venous catheter removed from patients by next generation sequencing: a preliminary clinical study
Article Snippet: .. For analysis of bacterial composition by NGS, the V3–V4 fragments of bacterial 16S rDNA were PCR-amplified, followed by index PCR and paired-end sequencing on an Illumina MiSeq device. ..

Purification:

Article Title: Ribosome maturation by the endoribonuclease YbeY stabilizes a type 3 secretion system transcript required for virulence of enterohemorrhagic Escherichia coli
Article Snippet: .. Linkers were ligated during purification, and cDNA libraries were synthesized from eluted RNA, PCR-amplified, and sequenced using the Illumina MiSeq platform. .. The YbeY binding profile was analyzed using the pyCRAC package , and statistically significant peaks were identified using pyCalculateFDR as described previously ( ) ( A ).

Sequencing:

Article Title: Partial bisulfite conversion for unique template sequencing
Article Snippet: .. After conversion, we sampled 4% from each converted sample and PCR-amplified (Figure ) using Illumina P5 and P7 sequencing adapters (for the complete conversion library, we sampled 40%). .. The resulting libraries were sequenced (Figure ) on an Illumina MiSeq (∼17 million paired-end reads per sample).

Article Title: Detection of bacterial DNA from central venous catheter removed from patients by next generation sequencing: a preliminary clinical study
Article Snippet: .. For analysis of bacterial composition by NGS, the V3–V4 fragments of bacterial 16S rDNA were PCR-amplified, followed by index PCR and paired-end sequencing on an Illumina MiSeq device. ..

Article Title: iCLIP Predicts the Dual Splicing Effects of TIA-RNA Interactions
Article Snippet: .. Linearised cDNA was then PCR-amplified using primers complementary to the adapter regions and subjected to high-throughput sequencing using Illumina GA2. .. Mapping Sequences to the Human Genome The sequences corresponding to the individual experiment were identified by their defined barcode, the random barcodes were registered, and the barcodes were removed before mapping the sequences to the human genome sequence (version Hg18/NCBI36), allowing one mismatch using Bowtie version 0.10.1 (command line: -a -m 1 -v 1).

Article Title: Influence of Heat Events on the Composition of Airborne Bacterial Communities in Urban Ecosystems
Article Snippet: .. PCR Amplification and Illumina Miseq PE300 Sequencing The V3–V4 regions of the 16S rRNA gene were PCR-amplified for Illumina Miseq PE300 sequencing. .. Amplicon PCR was performed in 25 μL volume reactions with 12.5 μL of 2× KAPA HiFi HotStart ReadyMix, 2.5 μL of microbial DNA, and 5 μL of each primer (Klindworth et al., 2013) using primers 341 F (CCTACGGGNGGCWGCAG) and 805 R (GACTACHVGGGTATCTAATCC).

Article Title: Next-generation DNA sequencing-based assay for measuring allelic expression imbalance (AEI) of candidate neuropsychiatric disorder genes in human brain
Article Snippet: .. Final steps in DNA sample preparation To prepare the DNA fragments for sequencing, the ligated gDNA- and cDNA-derived DNAs were independently PCR-amplified using Illumina Primers 1.1 and 1.2 and Phusion DNA polymerase (Finnzymes Oy). .. The PCR products were resolved by electrophoresis on 15% polyacrylamide gels and DNAs of the appropriate size extracted using QIAquick Gel Extraction Kits (Qiagen).

Polymerase Chain Reaction:

Article Title: Partial bisulfite conversion for unique template sequencing
Article Snippet: .. After conversion, we sampled 4% from each converted sample and PCR-amplified (Figure ) using Illumina P5 and P7 sequencing adapters (for the complete conversion library, we sampled 40%). .. The resulting libraries were sequenced (Figure ) on an Illumina MiSeq (∼17 million paired-end reads per sample).

Article Title: Detection of bacterial DNA from central venous catheter removed from patients by next generation sequencing: a preliminary clinical study
Article Snippet: .. For analysis of bacterial composition by NGS, the V3–V4 fragments of bacterial 16S rDNA were PCR-amplified, followed by index PCR and paired-end sequencing on an Illumina MiSeq device. ..

Article Title: Ribosome maturation by the endoribonuclease YbeY stabilizes a type 3 secretion system transcript required for virulence of enterohemorrhagic Escherichia coli
Article Snippet: .. Linkers were ligated during purification, and cDNA libraries were synthesized from eluted RNA, PCR-amplified, and sequenced using the Illumina MiSeq platform. .. The YbeY binding profile was analyzed using the pyCRAC package , and statistically significant peaks were identified using pyCalculateFDR as described previously ( ) ( A ).

Article Title: iCLIP Predicts the Dual Splicing Effects of TIA-RNA Interactions
Article Snippet: .. Linearised cDNA was then PCR-amplified using primers complementary to the adapter regions and subjected to high-throughput sequencing using Illumina GA2. .. Mapping Sequences to the Human Genome The sequences corresponding to the individual experiment were identified by their defined barcode, the random barcodes were registered, and the barcodes were removed before mapping the sequences to the human genome sequence (version Hg18/NCBI36), allowing one mismatch using Bowtie version 0.10.1 (command line: -a -m 1 -v 1).

Article Title: Influence of Heat Events on the Composition of Airborne Bacterial Communities in Urban Ecosystems
Article Snippet: .. PCR Amplification and Illumina Miseq PE300 Sequencing The V3–V4 regions of the 16S rRNA gene were PCR-amplified for Illumina Miseq PE300 sequencing. .. Amplicon PCR was performed in 25 μL volume reactions with 12.5 μL of 2× KAPA HiFi HotStart ReadyMix, 2.5 μL of microbial DNA, and 5 μL of each primer (Klindworth et al., 2013) using primers 341 F (CCTACGGGNGGCWGCAG) and 805 R (GACTACHVGGGTATCTAATCC).

Article Title: An ultraprocessive, accurate reverse transcriptase encoded by a metazoan group II intron
Article Snippet: .. Finally, the PCR-amplified products were pooled and samples for MarathonRT were sequenced on an Illumina Miseq sequencer in paired-end mode for 250 cycles (PE250) with 20% PhiX spike-in, whereas samples for SSIV and TGIRT were sequenced on an Illumina Hiseq sequencer in paired-end mode for 75 cycles (PE75) as 1% spike-in at YCGA. ..

Article Title: RIP-seq analysis of eukaryotic Sm proteins identifies three major categories of Sm-containing ribonucleoproteins
Article Snippet: .. Immunoprecipitated RNA was reverse transcribed to cDNA, fragmented, ligated with adapters, PCR-amplified and sequenced on an Illumina Genome Analyzer II. ..

Article Title: Next-generation DNA sequencing-based assay for measuring allelic expression imbalance (AEI) of candidate neuropsychiatric disorder genes in human brain
Article Snippet: .. Final steps in DNA sample preparation To prepare the DNA fragments for sequencing, the ligated gDNA- and cDNA-derived DNAs were independently PCR-amplified using Illumina Primers 1.1 and 1.2 and Phusion DNA polymerase (Finnzymes Oy). .. The PCR products were resolved by electrophoresis on 15% polyacrylamide gels and DNAs of the appropriate size extracted using QIAquick Gel Extraction Kits (Qiagen).

Immunoprecipitation:

Article Title: RIP-seq analysis of eukaryotic Sm proteins identifies three major categories of Sm-containing ribonucleoproteins
Article Snippet: .. Immunoprecipitated RNA was reverse transcribed to cDNA, fragmented, ligated with adapters, PCR-amplified and sequenced on an Illumina Genome Analyzer II. ..

High Throughput Screening Assay:

Article Title: iCLIP Predicts the Dual Splicing Effects of TIA-RNA Interactions
Article Snippet: .. Linearised cDNA was then PCR-amplified using primers complementary to the adapter regions and subjected to high-throughput sequencing using Illumina GA2. .. Mapping Sequences to the Human Genome The sequences corresponding to the individual experiment were identified by their defined barcode, the random barcodes were registered, and the barcodes were removed before mapping the sequences to the human genome sequence (version Hg18/NCBI36), allowing one mismatch using Bowtie version 0.10.1 (command line: -a -m 1 -v 1).

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    Illumina Inc pcr amplified illumina libraries
    'PER' genome-wide base composition bias curves . (a,b) Shown is the GC bias in <t>Illumina</t> reads from a 400-bp fragment library amplified using the standard <t>PCR</t> protocol (Phusion HF, short denaturation) on a fast-ramping thermocycler (red squares), Phusion HF with long denaturation and 2M betaine (black triangles), AccuPrime Taq HiFi with long denaturation and primer extension at 65°C (blue diamonds) or 60°C (purple diamonds). To calculate the observed to expected (unbiased) read coverage, the number of reads aligning to 50-bp windows at a given %GC was divided by the number of 50-bp windows that fall in this %GC category. This value was then normalized relative to the average value from 48% through 52% GC and plotted on a log 10 scale (a) or linear scale (b).
    Pcr Amplified Illumina Libraries, supplied by Illumina Inc, used in various techniques. Bioz Stars score: 85/100, based on 205 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    'PER' genome-wide base composition bias curves . (a,b) Shown is the GC bias in Illumina reads from a 400-bp fragment library amplified using the standard PCR protocol (Phusion HF, short denaturation) on a fast-ramping thermocycler (red squares), Phusion HF with long denaturation and 2M betaine (black triangles), AccuPrime Taq HiFi with long denaturation and primer extension at 65°C (blue diamonds) or 60°C (purple diamonds). To calculate the observed to expected (unbiased) read coverage, the number of reads aligning to 50-bp windows at a given %GC was divided by the number of 50-bp windows that fall in this %GC category. This value was then normalized relative to the average value from 48% through 52% GC and plotted on a log 10 scale (a) or linear scale (b).

    Journal: Genome Biology

    Article Title: Analyzing and minimizing PCR amplification bias in Illumina sequencing libraries

    doi: 10.1186/gb-2011-12-2-r18

    Figure Lengend Snippet: 'PER' genome-wide base composition bias curves . (a,b) Shown is the GC bias in Illumina reads from a 400-bp fragment library amplified using the standard PCR protocol (Phusion HF, short denaturation) on a fast-ramping thermocycler (red squares), Phusion HF with long denaturation and 2M betaine (black triangles), AccuPrime Taq HiFi with long denaturation and primer extension at 65°C (blue diamonds) or 60°C (purple diamonds). To calculate the observed to expected (unbiased) read coverage, the number of reads aligning to 50-bp windows at a given %GC was divided by the number of 50-bp windows that fall in this %GC category. This value was then normalized relative to the average value from 48% through 52% GC and plotted on a log 10 scale (a) or linear scale (b).

    Article Snippet: We identified the enrichment PCR step as the primary source of base-composition bias in fragment libraries and developed an optimized PCR protocol that produces libraries that are far less skewed than standard PCR-amplified Illumina libraries.

    Techniques: Genome Wide, Amplification, Polymerase Chain Reaction

    Sequencing bias with PCR-amplified and PCR-free libraries . (a,b) Shown is the mean normalized coverage of 50-bp windows in the human genome having the GC-content indicated on the x-axis for a PCR-free (orange dots) and a PCR-amplified (blue diamonds) Illumina sequencing library. Both fragment libraries had approximately 180-bp inserts. The PCR amplification was performed with AccuPrime Taq HiFi (long denat., primer extension at 65°C). The coverage was plotted on a log 10 (a) and a linear scale (b). The data points at extremely high GC, where the reads from the PCR-free library had a mean base quality of less than Q20 (open symbols), were omitted in the middle panel (b). (c) The ratios of the two curves in (a,b), that is, the fold-increase in mean coverage by sequencing a PCR-free library instead of a PCR-amplified library. The shaded histogram is the %GC distribution of 50-bp windows in the human genome. More than 99.9% of all 50-bp windows in the genome contain 8% to 88% GC and received a less than 1.25-fold increase in coverage. Less than 0.01% of all 50-bp windows contain 90% or more GC. The open circles at 96% and 98% GC denote data for which the mean base quality of the reads from the PCR-free library was below Q20.

    Journal: Genome Biology

    Article Title: Analyzing and minimizing PCR amplification bias in Illumina sequencing libraries

    doi: 10.1186/gb-2011-12-2-r18

    Figure Lengend Snippet: Sequencing bias with PCR-amplified and PCR-free libraries . (a,b) Shown is the mean normalized coverage of 50-bp windows in the human genome having the GC-content indicated on the x-axis for a PCR-free (orange dots) and a PCR-amplified (blue diamonds) Illumina sequencing library. Both fragment libraries had approximately 180-bp inserts. The PCR amplification was performed with AccuPrime Taq HiFi (long denat., primer extension at 65°C). The coverage was plotted on a log 10 (a) and a linear scale (b). The data points at extremely high GC, where the reads from the PCR-free library had a mean base quality of less than Q20 (open symbols), were omitted in the middle panel (b). (c) The ratios of the two curves in (a,b), that is, the fold-increase in mean coverage by sequencing a PCR-free library instead of a PCR-amplified library. The shaded histogram is the %GC distribution of 50-bp windows in the human genome. More than 99.9% of all 50-bp windows in the genome contain 8% to 88% GC and received a less than 1.25-fold increase in coverage. Less than 0.01% of all 50-bp windows contain 90% or more GC. The open circles at 96% and 98% GC denote data for which the mean base quality of the reads from the PCR-free library was below Q20.

    Article Snippet: We identified the enrichment PCR step as the primary source of base-composition bias in fragment libraries and developed an optimized PCR protocol that produces libraries that are far less skewed than standard PCR-amplified Illumina libraries.

    Techniques: Sequencing, Polymerase Chain Reaction, Amplification

    iCLIP identifies the TIA1 and TIAL1 crosslink sites with nucleotide resolution. Autoradiogram of 32 P-labelled RNA crosslinked to TIA1 (A) or TIAL1 (B) in HeLa cells. Immunoprecipitation was performed with either anti-TIA1 or anti-TIAL1 antibody using lysate from UV-crosslinked HeLa cells, cells transfected with TIA1 or TIAL1, TIA1/TIAL1 KD cells, or non-crosslinked cells. High and low RNase concentrations were used and protein G beads were used as a control. The Western blots below the autoradiograms show the input lysate used for each immunoprecipitation. (C) TIA1 and TIAL1 crosslink to uridine tracts downstream of the alternative 5′ splice sites in the CLIP4 gene. The cDNA positions are colour-coded for three replicate TIA1 and TIAL1 experiments, and the random barcode (shown on the left) is used to identify unique iCLIP cDNAs (number in brackets indicates the number of corresponding PCR duplicates). Below, the bar graphs show the cDNA count (number of cDNAs at each crosslink site). Pre-mRNA sequence is shown below with crosslink nucleotides in red. The exon and intron positions of the two isoforms of CLIP4 mRNA are shown at the bottom.

    Journal: PLoS Biology

    Article Title: iCLIP Predicts the Dual Splicing Effects of TIA-RNA Interactions

    doi: 10.1371/journal.pbio.1000530

    Figure Lengend Snippet: iCLIP identifies the TIA1 and TIAL1 crosslink sites with nucleotide resolution. Autoradiogram of 32 P-labelled RNA crosslinked to TIA1 (A) or TIAL1 (B) in HeLa cells. Immunoprecipitation was performed with either anti-TIA1 or anti-TIAL1 antibody using lysate from UV-crosslinked HeLa cells, cells transfected with TIA1 or TIAL1, TIA1/TIAL1 KD cells, or non-crosslinked cells. High and low RNase concentrations were used and protein G beads were used as a control. The Western blots below the autoradiograms show the input lysate used for each immunoprecipitation. (C) TIA1 and TIAL1 crosslink to uridine tracts downstream of the alternative 5′ splice sites in the CLIP4 gene. The cDNA positions are colour-coded for three replicate TIA1 and TIAL1 experiments, and the random barcode (shown on the left) is used to identify unique iCLIP cDNAs (number in brackets indicates the number of corresponding PCR duplicates). Below, the bar graphs show the cDNA count (number of cDNAs at each crosslink site). Pre-mRNA sequence is shown below with crosslink nucleotides in red. The exon and intron positions of the two isoforms of CLIP4 mRNA are shown at the bottom.

    Article Snippet: Linearised cDNA was then PCR-amplified using primers complementary to the adapter regions and subjected to high-throughput sequencing using Illumina GA2.

    Techniques: Immunoprecipitation, Transfection, Western Blot, Polymerase Chain Reaction, Sequencing

    Translation stabilizes the espD transcript. A , Northern blot analysis of espD ( left panel ) and recA ( right panel ) in WT ZAP193, Δ ybeY , and the ybeY -HTF strain backgrounds. B , transcription and translation of the first gene in the LEE4 polycistronic operon, sepL , was measured using GFP fusions. Left , the transcriptional sepL -GFP fusion (pCDR8) containing the native promoter and 8 nt of the sepL 5′-UTR was measured in ZAP193 and Δ ybeY backgrounds under T3S-permissive conditions. Right , the translational sepL -GFP fusion (pDW6) containing the native promoter and entire sepL CDS fused to GFP was similarly measured under T3S-permissive conditions. Error bars represent S.E. from biological triplicates. AU , arbitrary units. C , Western blotting for the T3S needle tip protein (EspD) in whole cells grown with and without a subinhibitory concentration of the translation initiation inhibitor kasugamycin (50 μg·ml −1 ). RecA was used as a loading control ( bottom panel ). D , RNA stability assay of espD and recA mRNAs with and without translating ribosomes. Transcription was blocked in EHEC str. ZAP193 by adding 1 mg·ml −1 rifampicin ( Rif ) ( closed circles ) or rifampicin and 1 mg·ml −1 kasugamycin ( Kas ) ( open circles ) at time 0. The relative abundance of mRNAs was monitored using quantitative RT-PCR. Error bars represent S.E. from biological triplicates. E , binding profiles for RNase E ( dark blue ), Hfq ( blue ), YbeY ( dark green ), and controls ( green ) are shown for transcripts from the LEE (ORFs are in gray ; espD is highlighted in red ). Protein binding to positive ( top ) and negative strands ( bottom ) of the transcriptome are shown. The polycistronic LEE1–5 transcripts are indicated above and below relevant ORFs.

    Journal: The Journal of Biological Chemistry

    Article Title: Ribosome maturation by the endoribonuclease YbeY stabilizes a type 3 secretion system transcript required for virulence of enterohemorrhagic Escherichia coli

    doi: 10.1074/jbc.RA117.000300

    Figure Lengend Snippet: Translation stabilizes the espD transcript. A , Northern blot analysis of espD ( left panel ) and recA ( right panel ) in WT ZAP193, Δ ybeY , and the ybeY -HTF strain backgrounds. B , transcription and translation of the first gene in the LEE4 polycistronic operon, sepL , was measured using GFP fusions. Left , the transcriptional sepL -GFP fusion (pCDR8) containing the native promoter and 8 nt of the sepL 5′-UTR was measured in ZAP193 and Δ ybeY backgrounds under T3S-permissive conditions. Right , the translational sepL -GFP fusion (pDW6) containing the native promoter and entire sepL CDS fused to GFP was similarly measured under T3S-permissive conditions. Error bars represent S.E. from biological triplicates. AU , arbitrary units. C , Western blotting for the T3S needle tip protein (EspD) in whole cells grown with and without a subinhibitory concentration of the translation initiation inhibitor kasugamycin (50 μg·ml −1 ). RecA was used as a loading control ( bottom panel ). D , RNA stability assay of espD and recA mRNAs with and without translating ribosomes. Transcription was blocked in EHEC str. ZAP193 by adding 1 mg·ml −1 rifampicin ( Rif ) ( closed circles ) or rifampicin and 1 mg·ml −1 kasugamycin ( Kas ) ( open circles ) at time 0. The relative abundance of mRNAs was monitored using quantitative RT-PCR. Error bars represent S.E. from biological triplicates. E , binding profiles for RNase E ( dark blue ), Hfq ( blue ), YbeY ( dark green ), and controls ( green ) are shown for transcripts from the LEE (ORFs are in gray ; espD is highlighted in red ). Protein binding to positive ( top ) and negative strands ( bottom ) of the transcriptome are shown. The polycistronic LEE1–5 transcripts are indicated above and below relevant ORFs.

    Article Snippet: Linkers were ligated during purification, and cDNA libraries were synthesized from eluted RNA, PCR-amplified, and sequenced using the Illumina MiSeq platform.

    Techniques: Northern Blot, Western Blot, Concentration Assay, Stability Assay, Quantitative RT-PCR, Binding Assay, Protein Binding

    Sm proteins associate with mature mRNAs. (a) Meta-gene analysis of read density around splice sites for all Drosophila and human Sm-associated intron-containing mRNAs in all RIP-seq experiments. (b) Meta-gene analysis of read density along the gene length for all Drosophila Sm-associated mRNAs quantified from oligodT and random hexamer primed libraries. (c) Example tracks for read density along the gene length for oligodT and random hexamer primed libraries. (d) Poly(A) tail length Sm-associated mRNAs (CG3997, CG1349 and CG3776) and non-associated mRNA (RpS2) from Y12 IP in S2 cells. IN, input total RNA; IP, immunoprecipitated RNA. The labels denote the length of poly(A) tails. Oligo(dT) 20 was used as the reverse primer for the reverse transcription and subsequent PCR, therefore producing the ‘smear’ of poly(A) tail. See Figure S11 in Additional file 1 for analysis of poly(A) containing reads for selected Sm-associated mRNAs.

    Journal: Genome Biology

    Article Title: RIP-seq analysis of eukaryotic Sm proteins identifies three major categories of Sm-containing ribonucleoproteins

    doi: 10.1186/gb-2014-15-1-r7

    Figure Lengend Snippet: Sm proteins associate with mature mRNAs. (a) Meta-gene analysis of read density around splice sites for all Drosophila and human Sm-associated intron-containing mRNAs in all RIP-seq experiments. (b) Meta-gene analysis of read density along the gene length for all Drosophila Sm-associated mRNAs quantified from oligodT and random hexamer primed libraries. (c) Example tracks for read density along the gene length for oligodT and random hexamer primed libraries. (d) Poly(A) tail length Sm-associated mRNAs (CG3997, CG1349 and CG3776) and non-associated mRNA (RpS2) from Y12 IP in S2 cells. IN, input total RNA; IP, immunoprecipitated RNA. The labels denote the length of poly(A) tails. Oligo(dT) 20 was used as the reverse primer for the reverse transcription and subsequent PCR, therefore producing the ‘smear’ of poly(A) tail. See Figure S11 in Additional file 1 for analysis of poly(A) containing reads for selected Sm-associated mRNAs.

    Article Snippet: Immunoprecipitated RNA was reverse transcribed to cDNA, fragmented, ligated with adapters, PCR-amplified and sequenced on an Illumina Genome Analyzer II.

    Techniques: Random Hexamer Labeling, Immunoprecipitation, Polymerase Chain Reaction

    RNA-Sm association is cell type-specific and not due to re-assortment. (a) RIP-qRT-PCR in da-Gal4 VFP-SmD1 fly ovary (anti-GFP) and S2 cells (Y12). Negative controls (Ctrl) used are 5S rRNA, Act5C and Smt3. CG9042 (Gapdh) is used as the normalization standard. snRNAs are shown separately due to the difference in scale. (b) mRNAs associated with Sm proteins in ovaries but not in S2 cells are expressed in S2 cells. t -Test for significance between IP and Ctrl: * P

    Journal: Genome Biology

    Article Title: RIP-seq analysis of eukaryotic Sm proteins identifies three major categories of Sm-containing ribonucleoproteins

    doi: 10.1186/gb-2014-15-1-r7

    Figure Lengend Snippet: RNA-Sm association is cell type-specific and not due to re-assortment. (a) RIP-qRT-PCR in da-Gal4 VFP-SmD1 fly ovary (anti-GFP) and S2 cells (Y12). Negative controls (Ctrl) used are 5S rRNA, Act5C and Smt3. CG9042 (Gapdh) is used as the normalization standard. snRNAs are shown separately due to the difference in scale. (b) mRNAs associated with Sm proteins in ovaries but not in S2 cells are expressed in S2 cells. t -Test for significance between IP and Ctrl: * P

    Article Snippet: Immunoprecipitated RNA was reverse transcribed to cDNA, fragmented, ligated with adapters, PCR-amplified and sequenced on an Illumina Genome Analyzer II.

    Techniques: Quantitative RT-PCR

    U1 snRNP binds mature mRNAs. (a) Putative base pairs between the 5′ end of U1 snRNA and the CG3776 mRNA coding region (upper panel). Within the putative region of base pairing, three translationally silent point mutations were introduced (bold blue letters) to disrupt the helix (lower panel). (b) Cartoon of the S2 cell transfection construct, showing the CG3776 expression unit. CG3776endo and CG3776tag indicate locations of primers for qRT-PCR. CG3776endo amplifies both endogenous and transfected CG3776 mRNAs, whereas CG3776tag amplifies transfected CG3776 mRNA only. The black star indicates the location of the putative U1 binding site. (c) pAW vector, pAW-CG3776wt and pAW-CG3776mut were transfected into S2 cells, and CG3776wt and CG3776mut expression was measured using qRT-PCR with the CG3776endo primer pair. GAPDH was used as normalization standard. (d) After pAW-CG3776wt and pAW-CG3776mut were transfected, anti-Sm (Y12) IPs were performed using S2 cell lysate. GAPDH was used as normalization standard. (e) Proposed model of snRNP-mRNA interactions. Distinct snRNPs (U1 and potentially others) associate with mature mRNAs via base pairing and/or protein-mediated interaction. Such interactions could serve as a platform to recruit RNA processing factors that act on multiple levels of RNA metabolism. t -Test for significance between IP and control (Ctrl): * P

    Journal: Genome Biology

    Article Title: RIP-seq analysis of eukaryotic Sm proteins identifies three major categories of Sm-containing ribonucleoproteins

    doi: 10.1186/gb-2014-15-1-r7

    Figure Lengend Snippet: U1 snRNP binds mature mRNAs. (a) Putative base pairs between the 5′ end of U1 snRNA and the CG3776 mRNA coding region (upper panel). Within the putative region of base pairing, three translationally silent point mutations were introduced (bold blue letters) to disrupt the helix (lower panel). (b) Cartoon of the S2 cell transfection construct, showing the CG3776 expression unit. CG3776endo and CG3776tag indicate locations of primers for qRT-PCR. CG3776endo amplifies both endogenous and transfected CG3776 mRNAs, whereas CG3776tag amplifies transfected CG3776 mRNA only. The black star indicates the location of the putative U1 binding site. (c) pAW vector, pAW-CG3776wt and pAW-CG3776mut were transfected into S2 cells, and CG3776wt and CG3776mut expression was measured using qRT-PCR with the CG3776endo primer pair. GAPDH was used as normalization standard. (d) After pAW-CG3776wt and pAW-CG3776mut were transfected, anti-Sm (Y12) IPs were performed using S2 cell lysate. GAPDH was used as normalization standard. (e) Proposed model of snRNP-mRNA interactions. Distinct snRNPs (U1 and potentially others) associate with mature mRNAs via base pairing and/or protein-mediated interaction. Such interactions could serve as a platform to recruit RNA processing factors that act on multiple levels of RNA metabolism. t -Test for significance between IP and control (Ctrl): * P

    Article Snippet: Immunoprecipitated RNA was reverse transcribed to cDNA, fragmented, ligated with adapters, PCR-amplified and sequenced on an Illumina Genome Analyzer II.

    Techniques: Transfection, Construct, Expressing, Quantitative RT-PCR, Binding Assay, Plasmid Preparation, Activated Clotting Time Assay