Structured Review

Illumina Inc oligo dt
Comparison or mRNA-seq libraries from <t>oligo-dT</t> and Cap-captured mRNA. A-B. Reads from dT or Cap-capture prepared libraries were aligned to sequences consisting of the 5’ or 3’ 25% of Refseq <t>mRNAs.</t> Reads aligning to the 5’ and 3’ portions of the transcript are plotted. Blue line indicates a ratio of one. C. Reads from mRNA-seq libraries prepared using oligo-dT captured mRNAs from mitotic and interphase Xenopus egg extract were aligned to the Xenopus laevis Unigene database. Relative abundance of each mRNA in mitotic and interphase extracts is plotted. Red points highlight two-fold differences between mitotic and interphase samples. D. Same experiment as in panel C except that the mRNAs were purified using Cap-capture prior to library preparation. Red points highlight two-fold differences. E. Scatterplot of mRNA abundance in oligo-dT-captured and Cap-captured mRNA libraries. F. The ratio of reads per mRNA in mitotic and interphase extracts are plotted for oligo-dT captured mRNAs (X-axis, data from panel A) and cap-captured mRNAs (Y-axis data from panel B). The colored points correspond to mRNAs with known changes in poly-A tail length (cdk1, green; Eg2/aurora-a, orange; mos, red; Xlcl1, yellow). Quadrants highlight two-fold differences between samples.
Oligo Dt, supplied by Illumina Inc, used in various techniques. Bioz Stars score: 91/100, based on 577 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/result/oligo dt/product/Illumina Inc
Average 91 stars, based on 577 article reviews
Price from $9.99 to $1999.99
oligo dt - by Bioz Stars, 2020-02
91/100 stars

Images

1) Product Images from "Combining Different mRNA Capture Methods to Analyze the Transcriptome: Analysis of the Xenopus laevis Transcriptome"

Article Title: Combining Different mRNA Capture Methods to Analyze the Transcriptome: Analysis of the Xenopus laevis Transcriptome

Journal: PLoS ONE

doi: 10.1371/journal.pone.0077700

Comparison or mRNA-seq libraries from oligo-dT and Cap-captured mRNA. A-B. Reads from dT or Cap-capture prepared libraries were aligned to sequences consisting of the 5’ or 3’ 25% of Refseq mRNAs. Reads aligning to the 5’ and 3’ portions of the transcript are plotted. Blue line indicates a ratio of one. C. Reads from mRNA-seq libraries prepared using oligo-dT captured mRNAs from mitotic and interphase Xenopus egg extract were aligned to the Xenopus laevis Unigene database. Relative abundance of each mRNA in mitotic and interphase extracts is plotted. Red points highlight two-fold differences between mitotic and interphase samples. D. Same experiment as in panel C except that the mRNAs were purified using Cap-capture prior to library preparation. Red points highlight two-fold differences. E. Scatterplot of mRNA abundance in oligo-dT-captured and Cap-captured mRNA libraries. F. The ratio of reads per mRNA in mitotic and interphase extracts are plotted for oligo-dT captured mRNAs (X-axis, data from panel A) and cap-captured mRNAs (Y-axis data from panel B). The colored points correspond to mRNAs with known changes in poly-A tail length (cdk1, green; Eg2/aurora-a, orange; mos, red; Xlcl1, yellow). Quadrants highlight two-fold differences between samples.
Figure Legend Snippet: Comparison or mRNA-seq libraries from oligo-dT and Cap-captured mRNA. A-B. Reads from dT or Cap-capture prepared libraries were aligned to sequences consisting of the 5’ or 3’ 25% of Refseq mRNAs. Reads aligning to the 5’ and 3’ portions of the transcript are plotted. Blue line indicates a ratio of one. C. Reads from mRNA-seq libraries prepared using oligo-dT captured mRNAs from mitotic and interphase Xenopus egg extract were aligned to the Xenopus laevis Unigene database. Relative abundance of each mRNA in mitotic and interphase extracts is plotted. Red points highlight two-fold differences between mitotic and interphase samples. D. Same experiment as in panel C except that the mRNAs were purified using Cap-capture prior to library preparation. Red points highlight two-fold differences. E. Scatterplot of mRNA abundance in oligo-dT-captured and Cap-captured mRNA libraries. F. The ratio of reads per mRNA in mitotic and interphase extracts are plotted for oligo-dT captured mRNAs (X-axis, data from panel A) and cap-captured mRNAs (Y-axis data from panel B). The colored points correspond to mRNAs with known changes in poly-A tail length (cdk1, green; Eg2/aurora-a, orange; mos, red; Xlcl1, yellow). Quadrants highlight two-fold differences between samples.

Techniques Used: Purification

Poly-A tail analysis of selected mRNAs. mRNAs that exhibited changes in Mitosis:Interphase (M:IF) abundance ratios in oligo-dT-captured samples, but not in Cap-captured samples were analyzed for poly-A tail length using the ePAT assay and anchored TVN reverse transcription controls. A. Six mRNAs with high M:IF ratios (aurora-a, esco2, fbox5, stx11, march7, and hexim1) showed longer poly-A tails in mitotic extract. Two mRNA (setd8 and MGC83922) with a low M:IF ratio showed very modest changes in poly-A tail lengths between Mitosis and Interphase. Red asterix indicates the position of the prominent TVN PCR product that is quantified in B. B. The amount of PCR product contained in the TVN-RT PCR reactions (red asterix in A) were quantified. The ratio of the amount of PCR product in Mitotic to Interphase extracts is presented in the first line. The ratio of each mRNA in Mitotic and Interphase extracts as determined by RNA-seq is presented below the PCR derived ratios for comparison. In addition five mRNAs with high M:IF ratios (aurora-1, fbox5, stx11, march7, and hexim1) had increased amounts of minimal poly-A tail PCR products in mitosis compared to interphase in TVN controls (quantified below gel) while both setd8 and MGC88922 had higher levels of TVN PCR products in interphase compared to mitotic extracts TVN PCR products indicate mRNAs with poly-A tails of 18 As. Line traces of ePAT PCR reactions presented in panel A. Black lines indicate traces from mitotic extract and red lines indicate traces from interaphse extract. D. Semi-quantitative PCR for each of the mRNAs tested in A was performed on RNA from mitotic and interphase extracts. Random hexamers were used to prime reverse transcription for these reactions. A second experiment showing very similar results is present in Figure S2.
Figure Legend Snippet: Poly-A tail analysis of selected mRNAs. mRNAs that exhibited changes in Mitosis:Interphase (M:IF) abundance ratios in oligo-dT-captured samples, but not in Cap-captured samples were analyzed for poly-A tail length using the ePAT assay and anchored TVN reverse transcription controls. A. Six mRNAs with high M:IF ratios (aurora-a, esco2, fbox5, stx11, march7, and hexim1) showed longer poly-A tails in mitotic extract. Two mRNA (setd8 and MGC83922) with a low M:IF ratio showed very modest changes in poly-A tail lengths between Mitosis and Interphase. Red asterix indicates the position of the prominent TVN PCR product that is quantified in B. B. The amount of PCR product contained in the TVN-RT PCR reactions (red asterix in A) were quantified. The ratio of the amount of PCR product in Mitotic to Interphase extracts is presented in the first line. The ratio of each mRNA in Mitotic and Interphase extracts as determined by RNA-seq is presented below the PCR derived ratios for comparison. In addition five mRNAs with high M:IF ratios (aurora-1, fbox5, stx11, march7, and hexim1) had increased amounts of minimal poly-A tail PCR products in mitosis compared to interphase in TVN controls (quantified below gel) while both setd8 and MGC88922 had higher levels of TVN PCR products in interphase compared to mitotic extracts TVN PCR products indicate mRNAs with poly-A tails of 18 As. Line traces of ePAT PCR reactions presented in panel A. Black lines indicate traces from mitotic extract and red lines indicate traces from interaphse extract. D. Semi-quantitative PCR for each of the mRNAs tested in A was performed on RNA from mitotic and interphase extracts. Random hexamers were used to prime reverse transcription for these reactions. A second experiment showing very similar results is present in Figure S2.

Techniques Used: Polymerase Chain Reaction, Reverse Transcription Polymerase Chain Reaction, RNA Sequencing Assay, Derivative Assay, Real-time Polymerase Chain Reaction

2) Product Images from "Quantification of massively parallel sequencing libraries – a comparative study of eight methods"

Article Title: Quantification of massively parallel sequencing libraries – a comparative study of eight methods

Journal: Scientific Reports

doi: 10.1038/s41598-018-19574-w

Quantification of synthetic double-stranded oligos. Four dilutions of two synthetic double-stranded oligos consisting of either the Ion Torrent “A” and “P1” adapter sequences ( a ) or the Illumina “i7” and “i5” adapter sequences ( b ) were quantified in duplicate with the NanoDrop ( ), Qubit ( ), Bioanalyzer ( ), GX Touch ( ), TapeStation ( ), and Fragment Analyzer ( ). The mean of the measured oligo concentrations were plotted against the concentrations given by the oligo supplier.
Figure Legend Snippet: Quantification of synthetic double-stranded oligos. Four dilutions of two synthetic double-stranded oligos consisting of either the Ion Torrent “A” and “P1” adapter sequences ( a ) or the Illumina “i7” and “i5” adapter sequences ( b ) were quantified in duplicate with the NanoDrop ( ), Qubit ( ), Bioanalyzer ( ), GX Touch ( ), TapeStation ( ), and Fragment Analyzer ( ). The mean of the measured oligo concentrations were plotted against the concentrations given by the oligo supplier.

Techniques Used:

3) Product Images from "Development and validation of a targeted gene sequencing panel for application to disparate cancers"

Article Title: Development and validation of a targeted gene sequencing panel for application to disparate cancers

Journal: Scientific Reports

doi: 10.1038/s41598-019-52000-3

Intragenic copy number detection and comparison to commercial panels. ( A ) Control genomic DNA samples were acquired from kConFab for sensitivity testing. Three of these samples included known exon duplications in BRCA1 , TP53 and MSH2 , which were assessed by the DeCON tool. Exons are numbered along the x-axis, and those of normal copy number are presented as blue dots. Amplifications are shown in red. The TP53 and AURKB genes are on opposing DNA strands hence the presence of the latter and its exons in this Figure. A similar genetic-overlap is observed for MSH2 to the left of the panel. ( B ) A commercially available pool of synthetic oligos against a normal genomic background was also obtained. Mutations were provided at variant allele frequencies (VAF) of 5–15% and 15–35%, or at germline frequencies. Presented are the number of detected and missed variants in our PV1 and PV2 panels relative to what was expected in AcroMetrix. This was compared to three other panels [AmpliSeq Cancer Hotspot Panel v2 (CHPv2), Illumina TruSeq Amplicon – Cancer Panel (TSCAP) and TruSight Tumor Panel 26 (TSTP)], the data for which were provided by the AcroMetrix manufacturer. Percent values on the right indicate the proportion of AcroMetrix variants actually targeted by the panels.
Figure Legend Snippet: Intragenic copy number detection and comparison to commercial panels. ( A ) Control genomic DNA samples were acquired from kConFab for sensitivity testing. Three of these samples included known exon duplications in BRCA1 , TP53 and MSH2 , which were assessed by the DeCON tool. Exons are numbered along the x-axis, and those of normal copy number are presented as blue dots. Amplifications are shown in red. The TP53 and AURKB genes are on opposing DNA strands hence the presence of the latter and its exons in this Figure. A similar genetic-overlap is observed for MSH2 to the left of the panel. ( B ) A commercially available pool of synthetic oligos against a normal genomic background was also obtained. Mutations were provided at variant allele frequencies (VAF) of 5–15% and 15–35%, or at germline frequencies. Presented are the number of detected and missed variants in our PV1 and PV2 panels relative to what was expected in AcroMetrix. This was compared to three other panels [AmpliSeq Cancer Hotspot Panel v2 (CHPv2), Illumina TruSeq Amplicon – Cancer Panel (TSCAP) and TruSight Tumor Panel 26 (TSTP)], the data for which were provided by the AcroMetrix manufacturer. Percent values on the right indicate the proportion of AcroMetrix variants actually targeted by the panels.

Techniques Used: Variant Assay, Amplification

4) Product Images from "Ribonuclease L and metal-ion-independent endoribonuclease cleavage sites in host and viral RNAs"

Article Title: Ribonuclease L and metal-ion-independent endoribonuclease cleavage sites in host and viral RNAs

Journal: Nucleic Acids Research

doi: 10.1093/nar/gku118

Viral RNA fragments produced by RNase L and RNase A. HCV and PV RNAs were incubated with RNase L and RNase A to produce RNA fragments for 2′, 3′-cyclic phosphate cDNA synthesis and sequencing. Agarose gel electrophoresis and ethidium bromide staining revealed the size of viral RNA fragments. ( A ) Diagram of HCV and PV RNAs. HCV RNA is 9648 bases long. PV RNA is 7500 bases long. ( B ) Viral RNAs incubated with RNase L. HCV and PV RNAs were incubated with RNase L for 20 min in the absence of 2-5A (no 2-5A), or with RNase L and 2-5A for 0, 2.5, 5, 10 and 20 min. ( C ) Viral RNAs incubated with RNase A. HCV and PV RNAs were incubated for 20 min in the absence of RNase A (−), and the presence of RNase A for 0, 2.5, 5, 10 and 20 min.
Figure Legend Snippet: Viral RNA fragments produced by RNase L and RNase A. HCV and PV RNAs were incubated with RNase L and RNase A to produce RNA fragments for 2′, 3′-cyclic phosphate cDNA synthesis and sequencing. Agarose gel electrophoresis and ethidium bromide staining revealed the size of viral RNA fragments. ( A ) Diagram of HCV and PV RNAs. HCV RNA is 9648 bases long. PV RNA is 7500 bases long. ( B ) Viral RNAs incubated with RNase L. HCV and PV RNAs were incubated with RNase L for 20 min in the absence of 2-5A (no 2-5A), or with RNase L and 2-5A for 0, 2.5, 5, 10 and 20 min. ( C ) Viral RNAs incubated with RNase A. HCV and PV RNAs were incubated for 20 min in the absence of RNase A (−), and the presence of RNase A for 0, 2.5, 5, 10 and 20 min.

Techniques Used: Produced, Incubation, Sequencing, Agarose Gel Electrophoresis, Staining

Cleavage sites mapped onto rRNA secondary and tertiary structures. Secondary and tertiary structures from Anger et al. ( 42 ). ( A ) RNase L cleavage sites in 18S rRNA secondary structure. Portion of 18S rRNA structure highlighting the location of RNase L cleavage sites. ( B ) Location of RNase L cleavage sites in 80S ribosome tertiary structure. RNase L-dependent cleavage sites highlighted in red spheres. ( C ) 3′-end of 5.8S and 5S rRNAs. 3′-end of 5.8S and 5S rRNAs highlighted in orange spheres. ( D ) RNase L-independent cleavage sites. Some representative RNase L-independent cleavage sites highlighted in yellow spheres.
Figure Legend Snippet: Cleavage sites mapped onto rRNA secondary and tertiary structures. Secondary and tertiary structures from Anger et al. ( 42 ). ( A ) RNase L cleavage sites in 18S rRNA secondary structure. Portion of 18S rRNA structure highlighting the location of RNase L cleavage sites. ( B ) Location of RNase L cleavage sites in 80S ribosome tertiary structure. RNase L-dependent cleavage sites highlighted in red spheres. ( C ) 3′-end of 5.8S and 5S rRNAs. 3′-end of 5.8S and 5S rRNAs highlighted in orange spheres. ( D ) RNase L-independent cleavage sites. Some representative RNase L-independent cleavage sites highlighted in yellow spheres.

Techniques Used:

Endoribonuclease cleavage sites in rRNAs from W12 HeLa cells. RNAs from mock-infected and PV-infected W12 HeLa cells ( Figure 3 B) were used for 2′, 3′-cyclic phosphate cDNA synthesis and Illumina sequencing. The location and frequency of cleavage sites in 28S rRNA ( A ), 18S rRNA ( B ), 5.8S rRNA ( C ) and 5S rRNA ( D ) are shown for mock-infected and PV-infected RNA samples isolated at 8 hpa. X-axis: Nucleotide position of each RNA. Y-axis: Percentage of total UMIs at each cleavage site. Dinucleotides at the 3′-end of abundant RNA fragments are annotated at the corresponding positions in the graphs. The locations of GC-rich expansion segments are highlighted by light blue rectangles. RNase L cleavage sites are highlighted in red.
Figure Legend Snippet: Endoribonuclease cleavage sites in rRNAs from W12 HeLa cells. RNAs from mock-infected and PV-infected W12 HeLa cells ( Figure 3 B) were used for 2′, 3′-cyclic phosphate cDNA synthesis and Illumina sequencing. The location and frequency of cleavage sites in 28S rRNA ( A ), 18S rRNA ( B ), 5.8S rRNA ( C ) and 5S rRNA ( D ) are shown for mock-infected and PV-infected RNA samples isolated at 8 hpa. X-axis: Nucleotide position of each RNA. Y-axis: Percentage of total UMIs at each cleavage site. Dinucleotides at the 3′-end of abundant RNA fragments are annotated at the corresponding positions in the graphs. The locations of GC-rich expansion segments are highlighted by light blue rectangles. RNase L cleavage sites are highlighted in red.

Techniques Used: Infection, Sequencing, Isolation

5) Product Images from "Ribonuclease L and metal-ion-independent endoribonuclease cleavage sites in host and viral RNAs"

Article Title: Ribonuclease L and metal-ion-independent endoribonuclease cleavage sites in host and viral RNAs

Journal: Nucleic Acids Research

doi: 10.1093/nar/gku118

Viral RNA fragments produced by RNase L and RNase A. HCV and PV RNAs were incubated with RNase L and RNase A to produce RNA fragments for 2′, 3′-cyclic phosphate cDNA synthesis and sequencing. Agarose gel electrophoresis and ethidium bromide staining revealed the size of viral RNA fragments. ( A ) Diagram of HCV and PV RNAs. HCV RNA is 9648 bases long. PV RNA is 7500 bases long. ( B ) Viral RNAs incubated with RNase L. HCV and PV RNAs were incubated with RNase L for 20 min in the absence of 2-5A (no 2-5A), or with RNase L and 2-5A for 0, 2.5, 5, 10 and 20 min. ( C ) Viral RNAs incubated with RNase A. HCV and PV RNAs were incubated for 20 min in the absence of RNase A (−), and the presence of RNase A for 0, 2.5, 5, 10 and 20 min.
Figure Legend Snippet: Viral RNA fragments produced by RNase L and RNase A. HCV and PV RNAs were incubated with RNase L and RNase A to produce RNA fragments for 2′, 3′-cyclic phosphate cDNA synthesis and sequencing. Agarose gel electrophoresis and ethidium bromide staining revealed the size of viral RNA fragments. ( A ) Diagram of HCV and PV RNAs. HCV RNA is 9648 bases long. PV RNA is 7500 bases long. ( B ) Viral RNAs incubated with RNase L. HCV and PV RNAs were incubated with RNase L for 20 min in the absence of 2-5A (no 2-5A), or with RNase L and 2-5A for 0, 2.5, 5, 10 and 20 min. ( C ) Viral RNAs incubated with RNase A. HCV and PV RNAs were incubated for 20 min in the absence of RNase A (−), and the presence of RNase A for 0, 2.5, 5, 10 and 20 min.

Techniques Used: Produced, Incubation, Sequencing, Agarose Gel Electrophoresis, Staining

Cleavage sites mapped onto rRNA secondary and tertiary structures. Secondary and tertiary structures from Anger et al. ( 42 ). ( A ) RNase L cleavage sites in 18S rRNA secondary structure. Portion of 18S rRNA structure highlighting the location of RNase L cleavage sites. ( B ) Location of RNase L cleavage sites in 80S ribosome tertiary structure. RNase L-dependent cleavage sites highlighted in red spheres. ( C ) 3′-end of 5.8S and 5S rRNAs. 3′-end of 5.8S and 5S rRNAs highlighted in orange spheres. ( D ) RNase L-independent cleavage sites. Some representative RNase L-independent cleavage sites highlighted in yellow spheres.
Figure Legend Snippet: Cleavage sites mapped onto rRNA secondary and tertiary structures. Secondary and tertiary structures from Anger et al. ( 42 ). ( A ) RNase L cleavage sites in 18S rRNA secondary structure. Portion of 18S rRNA structure highlighting the location of RNase L cleavage sites. ( B ) Location of RNase L cleavage sites in 80S ribosome tertiary structure. RNase L-dependent cleavage sites highlighted in red spheres. ( C ) 3′-end of 5.8S and 5S rRNAs. 3′-end of 5.8S and 5S rRNAs highlighted in orange spheres. ( D ) RNase L-independent cleavage sites. Some representative RNase L-independent cleavage sites highlighted in yellow spheres.

Techniques Used:

Endoribonuclease cleavage sites in rRNAs from W12 HeLa cells. RNAs from mock-infected and PV-infected W12 HeLa cells ( Figure 3 B) were used for 2′, 3′-cyclic phosphate cDNA synthesis and Illumina sequencing. The location and frequency of cleavage sites in 28S rRNA ( A ), 18S rRNA ( B ), 5.8S rRNA ( C ) and 5S rRNA ( D ) are shown for mock-infected and PV-infected RNA samples isolated at 8 hpa. X-axis: Nucleotide position of each RNA. Y-axis: Percentage of total UMIs at each cleavage site. Dinucleotides at the 3′-end of abundant RNA fragments are annotated at the corresponding positions in the graphs. The locations of GC-rich expansion segments are highlighted by light blue rectangles. RNase L cleavage sites are highlighted in red.
Figure Legend Snippet: Endoribonuclease cleavage sites in rRNAs from W12 HeLa cells. RNAs from mock-infected and PV-infected W12 HeLa cells ( Figure 3 B) were used for 2′, 3′-cyclic phosphate cDNA synthesis and Illumina sequencing. The location and frequency of cleavage sites in 28S rRNA ( A ), 18S rRNA ( B ), 5.8S rRNA ( C ) and 5S rRNA ( D ) are shown for mock-infected and PV-infected RNA samples isolated at 8 hpa. X-axis: Nucleotide position of each RNA. Y-axis: Percentage of total UMIs at each cleavage site. Dinucleotides at the 3′-end of abundant RNA fragments are annotated at the corresponding positions in the graphs. The locations of GC-rich expansion segments are highlighted by light blue rectangles. RNase L cleavage sites are highlighted in red.

Techniques Used: Infection, Sequencing, Isolation

6) Product Images from "Combining Different mRNA Capture Methods to Analyze the Transcriptome: Analysis of the Xenopus laevis Transcriptome"

Article Title: Combining Different mRNA Capture Methods to Analyze the Transcriptome: Analysis of the Xenopus laevis Transcriptome

Journal: PLoS ONE

doi: 10.1371/journal.pone.0077700

Comparison of de novo transcriptome assemblies. A. Transcripts assembled by the Abyss assembler from oligo-dT and Cap-captured mRNA libraries were aligned to the X. tropicalis ENSEMBL annotated transcripts using BlastX. The number of ENSEMBL transcripts that were matched by each assembler:library pair are represented as a Venn Diagram. The number of transcripts present in each assembler:library pair are listed in Table 2. Because multiple sequences from each de novo assembly align to X. tropicalis genes these numbers are omitted from the figure for the sake of simplicity. B. Same comparison as in A, except that Velvet was used as the assembler instead of Abyss. C. Transcripts assembled by Abyss or Velvet from Cap-capture mRNA libraries were aligned to the X. tropicalis ENSEMBL transcripts using BLAT. Unique and common ENSEMBL transcripts are represented by a Venn Diagram D. Same comparison as in C, except that dT libraries are compared instead of Cap-captured libraries. E-H. Fraction of each ENSEMBL transcript covered by transcripts assembled using indicated assembler:library pair (from A-D) was calculated.
Figure Legend Snippet: Comparison of de novo transcriptome assemblies. A. Transcripts assembled by the Abyss assembler from oligo-dT and Cap-captured mRNA libraries were aligned to the X. tropicalis ENSEMBL annotated transcripts using BlastX. The number of ENSEMBL transcripts that were matched by each assembler:library pair are represented as a Venn Diagram. The number of transcripts present in each assembler:library pair are listed in Table 2. Because multiple sequences from each de novo assembly align to X. tropicalis genes these numbers are omitted from the figure for the sake of simplicity. B. Same comparison as in A, except that Velvet was used as the assembler instead of Abyss. C. Transcripts assembled by Abyss or Velvet from Cap-capture mRNA libraries were aligned to the X. tropicalis ENSEMBL transcripts using BLAT. Unique and common ENSEMBL transcripts are represented by a Venn Diagram D. Same comparison as in C, except that dT libraries are compared instead of Cap-captured libraries. E-H. Fraction of each ENSEMBL transcript covered by transcripts assembled using indicated assembler:library pair (from A-D) was calculated.

Techniques Used:

Comparison or mRNA-seq libraries from oligo-dT and Cap-captured mRNA. A-B. Reads from dT or Cap-capture prepared libraries were aligned to sequences consisting of the 5’ or 3’ 25% of Refseq mRNAs. Reads aligning to the 5’ and 3’ portions of the transcript are plotted. Blue line indicates a ratio of one. C. Reads from mRNA-seq libraries prepared using oligo-dT captured mRNAs from mitotic and interphase Xenopus egg extract were aligned to the Xenopus laevis Unigene database. Relative abundance of each mRNA in mitotic and interphase extracts is plotted. Red points highlight two-fold differences between mitotic and interphase samples. D. Same experiment as in panel C except that the mRNAs were purified using Cap-capture prior to library preparation. Red points highlight two-fold differences. E. Scatterplot of mRNA abundance in oligo-dT-captured and Cap-captured mRNA libraries. F. The ratio of reads per mRNA in mitotic and interphase extracts are plotted for oligo-dT captured mRNAs (X-axis, data from panel A) and cap-captured mRNAs (Y-axis data from panel B). The colored points correspond to mRNAs with known changes in poly-A tail length (cdk1, green; Eg2/aurora-a, orange; mos, red; Xlcl1, yellow). Quadrants highlight two-fold differences between samples.
Figure Legend Snippet: Comparison or mRNA-seq libraries from oligo-dT and Cap-captured mRNA. A-B. Reads from dT or Cap-capture prepared libraries were aligned to sequences consisting of the 5’ or 3’ 25% of Refseq mRNAs. Reads aligning to the 5’ and 3’ portions of the transcript are plotted. Blue line indicates a ratio of one. C. Reads from mRNA-seq libraries prepared using oligo-dT captured mRNAs from mitotic and interphase Xenopus egg extract were aligned to the Xenopus laevis Unigene database. Relative abundance of each mRNA in mitotic and interphase extracts is plotted. Red points highlight two-fold differences between mitotic and interphase samples. D. Same experiment as in panel C except that the mRNAs were purified using Cap-capture prior to library preparation. Red points highlight two-fold differences. E. Scatterplot of mRNA abundance in oligo-dT-captured and Cap-captured mRNA libraries. F. The ratio of reads per mRNA in mitotic and interphase extracts are plotted for oligo-dT captured mRNAs (X-axis, data from panel A) and cap-captured mRNAs (Y-axis data from panel B). The colored points correspond to mRNAs with known changes in poly-A tail length (cdk1, green; Eg2/aurora-a, orange; mos, red; Xlcl1, yellow). Quadrants highlight two-fold differences between samples.

Techniques Used: Purification

Poly-A tail analysis of selected mRNAs. mRNAs that exhibited changes in Mitosis:Interphase (M:IF) abundance ratios in oligo-dT-captured samples, but not in Cap-captured samples were analyzed for poly-A tail length using the ePAT assay and anchored TVN reverse transcription controls. A. Six mRNAs with high M:IF ratios (aurora-a, esco2, fbox5, stx11, march7, and hexim1) showed longer poly-A tails in mitotic extract. Two mRNA (setd8 and MGC83922) with a low M:IF ratio showed very modest changes in poly-A tail lengths between Mitosis and Interphase. Red asterix indicates the position of the prominent TVN PCR product that is quantified in B. B. The amount of PCR product contained in the TVN-RT PCR reactions (red asterix in A) were quantified. The ratio of the amount of PCR product in Mitotic to Interphase extracts is presented in the first line. The ratio of each mRNA in Mitotic and Interphase extracts as determined by RNA-seq is presented below the PCR derived ratios for comparison. In addition five mRNAs with high M:IF ratios (aurora-1, fbox5, stx11, march7, and hexim1) had increased amounts of minimal poly-A tail PCR products in mitosis compared to interphase in TVN controls (quantified below gel) while both setd8 and MGC88922 had higher levels of TVN PCR products in interphase compared to mitotic extracts TVN PCR products indicate mRNAs with poly-A tails of 18 As. Line traces of ePAT PCR reactions presented in panel A. Black lines indicate traces from mitotic extract and red lines indicate traces from interaphse extract. D. Semi-quantitative PCR for each of the mRNAs tested in A was performed on RNA from mitotic and interphase extracts. Random hexamers were used to prime reverse transcription for these reactions. A second experiment showing very similar results is present in Figure S2.
Figure Legend Snippet: Poly-A tail analysis of selected mRNAs. mRNAs that exhibited changes in Mitosis:Interphase (M:IF) abundance ratios in oligo-dT-captured samples, but not in Cap-captured samples were analyzed for poly-A tail length using the ePAT assay and anchored TVN reverse transcription controls. A. Six mRNAs with high M:IF ratios (aurora-a, esco2, fbox5, stx11, march7, and hexim1) showed longer poly-A tails in mitotic extract. Two mRNA (setd8 and MGC83922) with a low M:IF ratio showed very modest changes in poly-A tail lengths between Mitosis and Interphase. Red asterix indicates the position of the prominent TVN PCR product that is quantified in B. B. The amount of PCR product contained in the TVN-RT PCR reactions (red asterix in A) were quantified. The ratio of the amount of PCR product in Mitotic to Interphase extracts is presented in the first line. The ratio of each mRNA in Mitotic and Interphase extracts as determined by RNA-seq is presented below the PCR derived ratios for comparison. In addition five mRNAs with high M:IF ratios (aurora-1, fbox5, stx11, march7, and hexim1) had increased amounts of minimal poly-A tail PCR products in mitosis compared to interphase in TVN controls (quantified below gel) while both setd8 and MGC88922 had higher levels of TVN PCR products in interphase compared to mitotic extracts TVN PCR products indicate mRNAs with poly-A tails of 18 As. Line traces of ePAT PCR reactions presented in panel A. Black lines indicate traces from mitotic extract and red lines indicate traces from interaphse extract. D. Semi-quantitative PCR for each of the mRNAs tested in A was performed on RNA from mitotic and interphase extracts. Random hexamers were used to prime reverse transcription for these reactions. A second experiment showing very similar results is present in Figure S2.

Techniques Used: Polymerase Chain Reaction, Reverse Transcription Polymerase Chain Reaction, RNA Sequencing Assay, Derivative Assay, Real-time Polymerase Chain Reaction

7) Product Images from "Quantification of massively parallel sequencing libraries – a comparative study of eight methods"

Article Title: Quantification of massively parallel sequencing libraries – a comparative study of eight methods

Journal: Scientific Reports

doi: 10.1038/s41598-018-19574-w

Quantification of synthetic double-stranded oligos. Four dilutions of two synthetic double-stranded oligos consisting of either the Ion Torrent “A” and “P1” adapter sequences ( a ) or the Illumina “i7” and “i5” adapter sequences ( b ) were quantified in duplicate with the NanoDrop ( ), Qubit ( ), Bioanalyzer ( ), GX Touch ( ), TapeStation ( ), and Fragment Analyzer ( ). The mean of the measured oligo concentrations were plotted against the concentrations given by the oligo supplier.
Figure Legend Snippet: Quantification of synthetic double-stranded oligos. Four dilutions of two synthetic double-stranded oligos consisting of either the Ion Torrent “A” and “P1” adapter sequences ( a ) or the Illumina “i7” and “i5” adapter sequences ( b ) were quantified in duplicate with the NanoDrop ( ), Qubit ( ), Bioanalyzer ( ), GX Touch ( ), TapeStation ( ), and Fragment Analyzer ( ). The mean of the measured oligo concentrations were plotted against the concentrations given by the oligo supplier.

Techniques Used:

8) Product Images from "Four Methods of Preparing mRNA 5? End Libraries Using the Illumina Sequencing Platform"

Article Title: Four Methods of Preparing mRNA 5? End Libraries Using the Illumina Sequencing Platform

Journal: PLoS ONE

doi: 10.1371/journal.pone.0101812

Library preparation using the CapSMART method. A) The protocol used either poly A+ (0.50–10 µg) or total (10–200 µg) RNA. B) De-phosphorylation of mono-, di-, and tri- phosphate groups from non-capped 5′ end molecules using alkaline phosphatase. C) Phosphorylation to add mono-phosphate to the non-capped 5′ end molecules using T4 Polynucleotide Kinase. D) Ligation of STOP oligos. A total of three kinds of oligonucleotides ( Table 2 : STOP1: iGiCiG, STOP2: iCiGiC, STOPMix: mixture of STOP1 and STOP2) were used in the present study. E) First-strand cDNA synthesis. F) Second-strand cDNA amplification by PCR with biotinylated 5′ end primers. G) Fragmentation of cDNA using a Bioruptor and collection of biotinylated 5′ ends using beads. H) Illumina sequencing library preparation.
Figure Legend Snippet: Library preparation using the CapSMART method. A) The protocol used either poly A+ (0.50–10 µg) or total (10–200 µg) RNA. B) De-phosphorylation of mono-, di-, and tri- phosphate groups from non-capped 5′ end molecules using alkaline phosphatase. C) Phosphorylation to add mono-phosphate to the non-capped 5′ end molecules using T4 Polynucleotide Kinase. D) Ligation of STOP oligos. A total of three kinds of oligonucleotides ( Table 2 : STOP1: iGiCiG, STOP2: iCiGiC, STOPMix: mixture of STOP1 and STOP2) were used in the present study. E) First-strand cDNA synthesis. F) Second-strand cDNA amplification by PCR with biotinylated 5′ end primers. G) Fragmentation of cDNA using a Bioruptor and collection of biotinylated 5′ ends using beads. H) Illumina sequencing library preparation.

Techniques Used: De-Phosphorylation Assay, Ligation, Amplification, Polymerase Chain Reaction, Sequencing

Frequency distribution of mapped sequence reads on the Drosophila melanogaster genome (Release 5) between nucleotide positions 4949000 and 4956000 on chromosome 2L. Only the sequences mapped on the minus strand are depicted. Gene locations ( Jon25Bi , Jon25Bii , Jon25Biii , and jet ) are depicted at the bottom of the figure. Plots from three libraries using SMART and ligation methods {adult (TG02, TG04, ID01, and ID02) and embryo RNA} and from three libraries using CapSMART and Non-CapSMART methods (ID01, ID02, ID03, ID04, ID05, and ID06) are depicted in the figure.
Figure Legend Snippet: Frequency distribution of mapped sequence reads on the Drosophila melanogaster genome (Release 5) between nucleotide positions 4949000 and 4956000 on chromosome 2L. Only the sequences mapped on the minus strand are depicted. Gene locations ( Jon25Bi , Jon25Bii , Jon25Biii , and jet ) are depicted at the bottom of the figure. Plots from three libraries using SMART and ligation methods {adult (TG02, TG04, ID01, and ID02) and embryo RNA} and from three libraries using CapSMART and Non-CapSMART methods (ID01, ID02, ID03, ID04, ID05, and ID06) are depicted in the figure.

Techniques Used: Sequencing, Ligation

Frequency distribution of mapped sequence reads on the Drosophila melanogaster genome (Release 5) between nucleotide positions 8041000 and 8044000 on chromosome 2L. Only the sequences mapped on the minus strand are depicted. Gene location ( RpL36A ) is depicted at the bottom of the figure. Plots from three libraries using SMART and ligation methods {adult (TG02, TG04, ID01, and ID02) and embryo RNA} and from three libraries using CapSMART and Non-CapSMART methods (ID01, ID02, ID03, ID04, ID05, and ID06) are depicted in the figure.
Figure Legend Snippet: Frequency distribution of mapped sequence reads on the Drosophila melanogaster genome (Release 5) between nucleotide positions 8041000 and 8044000 on chromosome 2L. Only the sequences mapped on the minus strand are depicted. Gene location ( RpL36A ) is depicted at the bottom of the figure. Plots from three libraries using SMART and ligation methods {adult (TG02, TG04, ID01, and ID02) and embryo RNA} and from three libraries using CapSMART and Non-CapSMART methods (ID01, ID02, ID03, ID04, ID05, and ID06) are depicted in the figure.

Techniques Used: Sequencing, Ligation

Frequency distribution of mapped sequence reads on the Drosophila melanogaster genome (Release 5) between nucleotide positions 7574000 and 7580000 on chromosome 2L. Only the sequences mapped on the minus strand are depicted. Gene locations ( Rapgap1 and CG13791 ) are depicted at the bottom of the figure. Plots from three libraries using SMART and ligation methods {adult (TG02, TG04, ID01, and ID02) and embryo RNA} and from three libraries using CapSMART and Non-CapSMART methods (ID01, ID02, ID03, ID04, ID05, and ID06) are depicted in the figure.
Figure Legend Snippet: Frequency distribution of mapped sequence reads on the Drosophila melanogaster genome (Release 5) between nucleotide positions 7574000 and 7580000 on chromosome 2L. Only the sequences mapped on the minus strand are depicted. Gene locations ( Rapgap1 and CG13791 ) are depicted at the bottom of the figure. Plots from three libraries using SMART and ligation methods {adult (TG02, TG04, ID01, and ID02) and embryo RNA} and from three libraries using CapSMART and Non-CapSMART methods (ID01, ID02, ID03, ID04, ID05, and ID06) are depicted in the figure.

Techniques Used: Sequencing, Ligation

Library preparation using the Non-CapSMART method. A) The protocol used either poly A+ (0.50–10 µg) or total (10–200 µg) RNA. B) De-phosphorylation of mono-, di-, and tri- phosphate groups from non-capped 5′ end molecules using alkaline phosphatase. C) Tobacco Acid Pyrophosphatase treatment to remove the 5′ cap structure, exposing a mono-phosphate group for subsequent ligation. D) Ligation of STOP oligos. A total of three kinds of oligonucleotides ( Table 2 : STOP1: iGiCiG, STOP2: iCiGiC, STOPMix: mixture of STOP1 and STOP2) were used in the present study. E) First-strand cDNA synthesis. F) Second-strand cDNA amplification by PCR with biotinylated 5′ end primers. G) Fragmentation of cDNA using a Bioruptor and collection of biotinylated 5′ ends using beads. H) Illumina sequencing library preparation.
Figure Legend Snippet: Library preparation using the Non-CapSMART method. A) The protocol used either poly A+ (0.50–10 µg) or total (10–200 µg) RNA. B) De-phosphorylation of mono-, di-, and tri- phosphate groups from non-capped 5′ end molecules using alkaline phosphatase. C) Tobacco Acid Pyrophosphatase treatment to remove the 5′ cap structure, exposing a mono-phosphate group for subsequent ligation. D) Ligation of STOP oligos. A total of three kinds of oligonucleotides ( Table 2 : STOP1: iGiCiG, STOP2: iCiGiC, STOPMix: mixture of STOP1 and STOP2) were used in the present study. E) First-strand cDNA synthesis. F) Second-strand cDNA amplification by PCR with biotinylated 5′ end primers. G) Fragmentation of cDNA using a Bioruptor and collection of biotinylated 5′ ends using beads. H) Illumina sequencing library preparation.

Techniques Used: De-Phosphorylation Assay, Ligation, Amplification, Polymerase Chain Reaction, Sequencing

9) Product Images from "Massive transcriptional start site analysis of human genes in hypoxia cells"

Article Title: Massive transcriptional start site analysis of human genes in hypoxia cells

Journal: Nucleic Acids Research

doi: 10.1093/nar/gkp066

Hypoxia-induced TSCs for putatively non-protein-coding RNAs. ( A ) Genomic positions of the regions in which activated TSSs highly concentrated (red circle). Number of RefSeq genes overlapping the corresponding 100 kb region is shown in the left margin. Examples of regions in which large numbers of transcription initiation sites were induced by hypoxia even in intergenic regions [( B) : a 100 kb region in Chromosome 8] and inside genic regions [( C ): a 100 kb region in Chromosome 17]. The vertical axis represents fold induction of the TSS-tag counts. TSSs of the genic region of NM_019‱613 (WDR45-like protein gene) are shown in the bottom margin. The direction of the transcription of the RefSeq gene is represented by a red arrow. Radius of each circle represents the number of TSS-tags. Colour of each circle indicates the direction of the transcription (red: same direction with the RefSeq gene; blue: opposite direction of the RefSeq gene). Putative alternative promoters on which confirmation analysis by real-time RT–PCR is shown in ( D ) are indicated in red and blue letters (AP1-3). AP: Alternative Promoter. (D) Real-time RT–PCR analysis of the putative alternative promoters, AP1-3, shown in (C). Fold inductions calculated by TSS-tag counts and real-time RT–PCR are shown in the third and fourth column, respectively. Primer sequences are shown in Supplementary Table 10 . Note that we used first strand single-strand cDNA as template, so that the PCR amplification should be strand-sensitive. (*) Also note that fold inductions estimated for AP3 by TSS-tag counts were the sum of the upstream promoters. As the AP3 were located inside of the last exon, it was impossible to design PCR primers which discriminate the transcript products of AP3 from those of other upstream promoters. Results of the independent oligo-cap RACE analysis for each of the APs are also shown in Supplementary Figure 6 .
Figure Legend Snippet: Hypoxia-induced TSCs for putatively non-protein-coding RNAs. ( A ) Genomic positions of the regions in which activated TSSs highly concentrated (red circle). Number of RefSeq genes overlapping the corresponding 100 kb region is shown in the left margin. Examples of regions in which large numbers of transcription initiation sites were induced by hypoxia even in intergenic regions [( B) : a 100 kb region in Chromosome 8] and inside genic regions [( C ): a 100 kb region in Chromosome 17]. The vertical axis represents fold induction of the TSS-tag counts. TSSs of the genic region of NM_019‱613 (WDR45-like protein gene) are shown in the bottom margin. The direction of the transcription of the RefSeq gene is represented by a red arrow. Radius of each circle represents the number of TSS-tags. Colour of each circle indicates the direction of the transcription (red: same direction with the RefSeq gene; blue: opposite direction of the RefSeq gene). Putative alternative promoters on which confirmation analysis by real-time RT–PCR is shown in ( D ) are indicated in red and blue letters (AP1-3). AP: Alternative Promoter. (D) Real-time RT–PCR analysis of the putative alternative promoters, AP1-3, shown in (C). Fold inductions calculated by TSS-tag counts and real-time RT–PCR are shown in the third and fourth column, respectively. Primer sequences are shown in Supplementary Table 10 . Note that we used first strand single-strand cDNA as template, so that the PCR amplification should be strand-sensitive. (*) Also note that fold inductions estimated for AP3 by TSS-tag counts were the sum of the upstream promoters. As the AP3 were located inside of the last exon, it was impossible to design PCR primers which discriminate the transcript products of AP3 from those of other upstream promoters. Results of the independent oligo-cap RACE analysis for each of the APs are also shown in Supplementary Figure 6 .

Techniques Used: Quantitative RT-PCR, Polymerase Chain Reaction, Amplification

Validation analyses of the TSS-tag library. ( A ) Mapped positions of the TSS-tags relative to the RefSeq genes were evaluated. Population of the TSS-tags mapped at the corresponding positions indicated by the color bars in the margin is shown. The right circle graph shows the composition of the blue section in the left circle graph. ( B ) Distribution of the luciferase activities of the upstream 1 kb regions of the TSSs ( n = 351; right). Luciferase activities of upstream regions of the TSS-tags that were not supported by any RefSeq gene models are calculated separately ( n = 20; left). Luciferase activities were normalized against the average luciferase activity of randomly isolated 1 kb genomic fragments ( n = 251). For further details, see the reference ( 27 ). ( C ) Correlation between the TSS-tag counts and the copy number estimated by real-time RT–PCR normalized by individual plasmids ( n = 105). Each value is the average of three experiments. Sequences of the used primers and quantitative data are presented in Supplementary Table 10 . R: correlation-coefficient calculated by linear regression. Note that, because the graph is written in log scale and the y intersect is not 0, the liner regression line is curved where the x value is small. ( D ) Examples of the real-time RT–PCR and independent oligo-cap RACE analyses. Experimental conditions are shown in the margin. For details, see Materials and methods section. For the primer, ‘Tag’ indicates the PCR primer targeted to the overlapping region of the TSS-tag and ‘RACE’ indicates the PCR primer targeted to the cap-replacing oligo. APID: alternative promoter ID. M: molecular marker.
Figure Legend Snippet: Validation analyses of the TSS-tag library. ( A ) Mapped positions of the TSS-tags relative to the RefSeq genes were evaluated. Population of the TSS-tags mapped at the corresponding positions indicated by the color bars in the margin is shown. The right circle graph shows the composition of the blue section in the left circle graph. ( B ) Distribution of the luciferase activities of the upstream 1 kb regions of the TSSs ( n = 351; right). Luciferase activities of upstream regions of the TSS-tags that were not supported by any RefSeq gene models are calculated separately ( n = 20; left). Luciferase activities were normalized against the average luciferase activity of randomly isolated 1 kb genomic fragments ( n = 251). For further details, see the reference ( 27 ). ( C ) Correlation between the TSS-tag counts and the copy number estimated by real-time RT–PCR normalized by individual plasmids ( n = 105). Each value is the average of three experiments. Sequences of the used primers and quantitative data are presented in Supplementary Table 10 . R: correlation-coefficient calculated by linear regression. Note that, because the graph is written in log scale and the y intersect is not 0, the liner regression line is curved where the x value is small. ( D ) Examples of the real-time RT–PCR and independent oligo-cap RACE analyses. Experimental conditions are shown in the margin. For details, see Materials and methods section. For the primer, ‘Tag’ indicates the PCR primer targeted to the overlapping region of the TSS-tag and ‘RACE’ indicates the PCR primer targeted to the cap-replacing oligo. APID: alternative promoter ID. M: molecular marker.

Techniques Used: Luciferase, Activity Assay, Isolation, Quantitative RT-PCR, Polymerase Chain Reaction, Marker

10) Product Images from "Ribonuclease L and metal-ion-independent endoribonuclease cleavage sites in host and viral RNAs"

Article Title: Ribonuclease L and metal-ion-independent endoribonuclease cleavage sites in host and viral RNAs

Journal: Nucleic Acids Research

doi: 10.1093/nar/gku118

Viral RNA fragments produced by RNase L and RNase A. HCV and PV RNAs were incubated with RNase L and RNase A to produce RNA fragments for 2′, 3′-cyclic phosphate cDNA synthesis and sequencing. Agarose gel electrophoresis and ethidium bromide staining revealed the size of viral RNA fragments. ( A ) Diagram of HCV and PV RNAs. HCV RNA is 9648 bases long. PV RNA is 7500 bases long. ( B ) Viral RNAs incubated with RNase L. HCV and PV RNAs were incubated with RNase L for 20 min in the absence of 2-5A (no 2-5A), or with RNase L and 2-5A for 0, 2.5, 5, 10 and 20 min. ( C ) Viral RNAs incubated with RNase A. HCV and PV RNAs were incubated for 20 min in the absence of RNase A (−), and the presence of RNase A for 0, 2.5, 5, 10 and 20 min.
Figure Legend Snippet: Viral RNA fragments produced by RNase L and RNase A. HCV and PV RNAs were incubated with RNase L and RNase A to produce RNA fragments for 2′, 3′-cyclic phosphate cDNA synthesis and sequencing. Agarose gel electrophoresis and ethidium bromide staining revealed the size of viral RNA fragments. ( A ) Diagram of HCV and PV RNAs. HCV RNA is 9648 bases long. PV RNA is 7500 bases long. ( B ) Viral RNAs incubated with RNase L. HCV and PV RNAs were incubated with RNase L for 20 min in the absence of 2-5A (no 2-5A), or with RNase L and 2-5A for 0, 2.5, 5, 10 and 20 min. ( C ) Viral RNAs incubated with RNase A. HCV and PV RNAs were incubated for 20 min in the absence of RNase A (−), and the presence of RNase A for 0, 2.5, 5, 10 and 20 min.

Techniques Used: Produced, Incubation, Sequencing, Agarose Gel Electrophoresis, Staining

Cleavage sites mapped onto rRNA secondary and tertiary structures. Secondary and tertiary structures from Anger et al. ( 42 ). ( A ) RNase L cleavage sites in 18S rRNA secondary structure. Portion of 18S rRNA structure highlighting the location of RNase L cleavage sites. ( B ) Location of RNase L cleavage sites in 80S ribosome tertiary structure. RNase L-dependent cleavage sites highlighted in red spheres. ( C ) 3′-end of 5.8S and 5S rRNAs. 3′-end of 5.8S and 5S rRNAs highlighted in orange spheres. ( D ) RNase L-independent cleavage sites. Some representative RNase L-independent cleavage sites highlighted in yellow spheres.
Figure Legend Snippet: Cleavage sites mapped onto rRNA secondary and tertiary structures. Secondary and tertiary structures from Anger et al. ( 42 ). ( A ) RNase L cleavage sites in 18S rRNA secondary structure. Portion of 18S rRNA structure highlighting the location of RNase L cleavage sites. ( B ) Location of RNase L cleavage sites in 80S ribosome tertiary structure. RNase L-dependent cleavage sites highlighted in red spheres. ( C ) 3′-end of 5.8S and 5S rRNAs. 3′-end of 5.8S and 5S rRNAs highlighted in orange spheres. ( D ) RNase L-independent cleavage sites. Some representative RNase L-independent cleavage sites highlighted in yellow spheres.

Techniques Used:

Endoribonuclease cleavage sites in rRNAs from W12 HeLa cells. RNAs from mock-infected and PV-infected W12 HeLa cells ( Figure 3 B) were used for 2′, 3′-cyclic phosphate cDNA synthesis and Illumina sequencing. The location and frequency of cleavage sites in 28S rRNA ( A ), 18S rRNA ( B ), 5.8S rRNA ( C ) and 5S rRNA ( D ) are shown for mock-infected and PV-infected RNA samples isolated at 8 hpa. X-axis: Nucleotide position of each RNA. Y-axis: Percentage of total UMIs at each cleavage site. Dinucleotides at the 3′-end of abundant RNA fragments are annotated at the corresponding positions in the graphs. The locations of GC-rich expansion segments are highlighted by light blue rectangles. RNase L cleavage sites are highlighted in red.
Figure Legend Snippet: Endoribonuclease cleavage sites in rRNAs from W12 HeLa cells. RNAs from mock-infected and PV-infected W12 HeLa cells ( Figure 3 B) were used for 2′, 3′-cyclic phosphate cDNA synthesis and Illumina sequencing. The location and frequency of cleavage sites in 28S rRNA ( A ), 18S rRNA ( B ), 5.8S rRNA ( C ) and 5S rRNA ( D ) are shown for mock-infected and PV-infected RNA samples isolated at 8 hpa. X-axis: Nucleotide position of each RNA. Y-axis: Percentage of total UMIs at each cleavage site. Dinucleotides at the 3′-end of abundant RNA fragments are annotated at the corresponding positions in the graphs. The locations of GC-rich expansion segments are highlighted by light blue rectangles. RNase L cleavage sites are highlighted in red.

Techniques Used: Infection, Sequencing, Isolation

11) Product Images from "Transcriptome profiling of mouse brains with qkI-deficient oligodendrocytes reveals major alternative splicing defects including self-splicing"

Article Title: Transcriptome profiling of mouse brains with qkI-deficient oligodendrocytes reveals major alternative splicing defects including self-splicing

Journal: Scientific Reports

doi: 10.1038/s41598-017-06211-1

Alternative splicing event in Bcas1, Sema6a and Capzb in QKI FL / FL ; Olig2 - Cre and QKI FL / FL ;- mice. ( a , b , c ) Left: IGV views of the exon skipping events in Bcas1 , Sema6a and Capzb . All samples are represented on the same scale for each screenshot. Black arrows represent the primers used for validation by RT-PCR. Right: Validation of exon skipping events by semi-quantitative PCR in n = 3 mice per genotype. The constitutive exon is present in all isoforms of the gene and is not alternatively spliced. The pixel densities of PCR bands from 3 independent mice brain RNA samples were quantified using ImageJ software, normalized over wild-type mice and represented as mean ± SEM (Student t -test, **p
Figure Legend Snippet: Alternative splicing event in Bcas1, Sema6a and Capzb in QKI FL / FL ; Olig2 - Cre and QKI FL / FL ;- mice. ( a , b , c ) Left: IGV views of the exon skipping events in Bcas1 , Sema6a and Capzb . All samples are represented on the same scale for each screenshot. Black arrows represent the primers used for validation by RT-PCR. Right: Validation of exon skipping events by semi-quantitative PCR in n = 3 mice per genotype. The constitutive exon is present in all isoforms of the gene and is not alternatively spliced. The pixel densities of PCR bands from 3 independent mice brain RNA samples were quantified using ImageJ software, normalized over wild-type mice and represented as mean ± SEM (Student t -test, **p

Techniques Used: Mouse Assay, Reverse Transcription Polymerase Chain Reaction, Real-time Polymerase Chain Reaction, Polymerase Chain Reaction, Software

Gene ontology analysis. Gene Ontology analysis shows enriched Biological Processes in the lists of upregulated genes (a) and downregulated genes (b) in QKI FL/FL;Olig2-Cre mice. Genes with adjusted p-value
Figure Legend Snippet: Gene ontology analysis. Gene Ontology analysis shows enriched Biological Processes in the lists of upregulated genes (a) and downregulated genes (b) in QKI FL/FL;Olig2-Cre mice. Genes with adjusted p-value

Techniques Used: Mouse Assay

Brain alternative splicing patterns of mice with QKI-deficient oligodendrocytes. ( a ) Types of alternative splicing events identified in QKI FL / FL ; Olig2 - Cre mice. For skipped exons (SE), the type of exon affected is also indicated. Exons were considered to be “cassette exons” if they were not the first or last exon in any of the gene isoforms or “mixed exons” if they were not the first or last exon in at least one isoform. Abbreviations: SE: skipped exon, A5SS: alternative 5′ splice site, A3SS: alternative 3′ splice site, RI: retained intron. ( b , c ) Motif enrichment analysis. Percentage of sequences with the motif ACUAA in introns neighboring SE events compared to a set of background sequences for the stringent SE candidates called by both methods (b) or for all SE candidates ( c ). The occurrence of the motif was counted in a random set of non-alternatively spliced control sequences of the same size of the set of SE events. This was repeated 1,000 times to get the distribution of the motif occurrence in control sequences. The p-value corresponds to the empirical p-value of the enrichment for the set of SE events compared to the control distribution. (d) Gene Ontology analysis shows enriched molecular function, biological processes and cellular component in the list of genes where the stringent alternative splicing events were found. For Molecular Function, all GO terms with q-value
Figure Legend Snippet: Brain alternative splicing patterns of mice with QKI-deficient oligodendrocytes. ( a ) Types of alternative splicing events identified in QKI FL / FL ; Olig2 - Cre mice. For skipped exons (SE), the type of exon affected is also indicated. Exons were considered to be “cassette exons” if they were not the first or last exon in any of the gene isoforms or “mixed exons” if they were not the first or last exon in at least one isoform. Abbreviations: SE: skipped exon, A5SS: alternative 5′ splice site, A3SS: alternative 3′ splice site, RI: retained intron. ( b , c ) Motif enrichment analysis. Percentage of sequences with the motif ACUAA in introns neighboring SE events compared to a set of background sequences for the stringent SE candidates called by both methods (b) or for all SE candidates ( c ). The occurrence of the motif was counted in a random set of non-alternatively spliced control sequences of the same size of the set of SE events. This was repeated 1,000 times to get the distribution of the motif occurrence in control sequences. The p-value corresponds to the empirical p-value of the enrichment for the set of SE events compared to the control distribution. (d) Gene Ontology analysis shows enriched molecular function, biological processes and cellular component in the list of genes where the stringent alternative splicing events were found. For Molecular Function, all GO terms with q-value

Techniques Used: Mouse Assay

Alterative splicing patterns of qkI in QKI FL / FL ; Olig2 - Cre and QKI FL / FL ;- mice. ( a ) IGV view of the alternative splicing events in qkI . All samples are represented on the same scale for each screenshot. ( b ) A schematic of the 3′-UTR of mouse qkI gene showing the location of Quaking Response Elements (QREs) identified. QREs are either a perfect match to consensus QRE or differ by one mismatch.
Figure Legend Snippet: Alterative splicing patterns of qkI in QKI FL / FL ; Olig2 - Cre and QKI FL / FL ;- mice. ( a ) IGV view of the alternative splicing events in qkI . All samples are represented on the same scale for each screenshot. ( b ) A schematic of the 3′-UTR of mouse qkI gene showing the location of Quaking Response Elements (QREs) identified. QREs are either a perfect match to consensus QRE or differ by one mismatch.

Techniques Used: Mouse Assay

Gene expression profiles comparing brains of QKI FL / FL ; Olig2 - Cre and QKI FL / FL ;- mice. ( a ) Global effects of  QKI FL/FL;Olig2-Cre  on cells were evaluated by unsupervised clustering of samples based on expression profiles of 1,000 most variant genes. QKI FL/FL;Olig2-Cre and QKI FL/FL;- mice form robust, distinct clusters, both with hierarchical clustering (right) and Principal Component Analysis (left). ( b ) Volcano plot representing the results of differential expression analysis. Genes with adjusted p-value
Figure Legend Snippet: Gene expression profiles comparing brains of QKI FL / FL ; Olig2 - Cre and QKI FL / FL ;- mice. ( a ) Global effects of  QKI FL/FL;Olig2-Cre  on cells were evaluated by unsupervised clustering of samples based on expression profiles of 1,000 most variant genes. QKI FL/FL;Olig2-Cre and QKI FL/FL;- mice form robust, distinct clusters, both with hierarchical clustering (right) and Principal Component Analysis (left). ( b ) Volcano plot representing the results of differential expression analysis. Genes with adjusted p-value

Techniques Used: Expressing, Mouse Assay, Variant Assay

12) Product Images from "Investigation of Experimental Factors That Underlie BRCA1/2 mRNA Isoform Expression Variation: Recommendations for Utilizing Targeted RNA Sequencing to Evaluate Potential Spliceogenic Variants"

Article Title: Investigation of Experimental Factors That Underlie BRCA1/2 mRNA Isoform Expression Variation: Recommendations for Utilizing Targeted RNA Sequencing to Evaluate Potential Spliceogenic Variants

Journal: Frontiers in Oncology

doi: 10.3389/fonc.2018.00140

Relative expression of BRCA1 and BRCA2 mRNA isoforms in rare variant samples compared to controls. (A) Natural expression ranges of mRNA splice isoforms calculated from lymphoblastoid cell lines (LCLs) not containing any known spliceogenic variants in BRCA1 (A) and BRCA2 (C) . Colored symbols overlaid indicate the relative mRNA isoform expression in LCLs containing known BRCA1 (B) or BRCA2 (D) splice disrupting variants. Only mRNA splice isoforms that were detected by more than 10 reads in at least two controls were included. Mean and upper and lower limits shown for each isoform [SE (95%)].
Figure Legend Snippet: Relative expression of BRCA1 and BRCA2 mRNA isoforms in rare variant samples compared to controls. (A) Natural expression ranges of mRNA splice isoforms calculated from lymphoblastoid cell lines (LCLs) not containing any known spliceogenic variants in BRCA1 (A) and BRCA2 (C) . Colored symbols overlaid indicate the relative mRNA isoform expression in LCLs containing known BRCA1 (B) or BRCA2 (D) splice disrupting variants. Only mRNA splice isoforms that were detected by more than 10 reads in at least two controls were included. Mean and upper and lower limits shown for each isoform [SE (95%)].

Techniques Used: Expressing, Variant Assay

BRCA2 mRNA isoforms detected at six time points in an lymphoblastoid cell line (sample #7, Table S1 in Supplementary Material) treated with an nonsense-mediated decay inhibitor. A freeze–thaw process was undertaken after time points two and four. Three technical replicates are listed under each time point.
Figure Legend Snippet: BRCA2 mRNA isoforms detected at six time points in an lymphoblastoid cell line (sample #7, Table S1 in Supplementary Material) treated with an nonsense-mediated decay inhibitor. A freeze–thaw process was undertaken after time points two and four. Three technical replicates are listed under each time point.

Techniques Used:

13) Product Images from "High‐throughput identification of RNA nuclear enrichment sequences"

Article Title: High‐throughput identification of RNA nuclear enrichment sequences

Journal: The EMBO Journal

doi: 10.15252/embj.201798452

A Massively Parallel RNA Assay ( MPRNA ) to identify RNA nuclear enrichment signals Experimental overview. Far left : oligonucleotide pool design. Double‐stranded DNA (dsDNA) oligonucleotides were designed by computationally scanning 38 parental lncRNA transcripts ( Table EV1 ) in 110‐nt windows, with 10‐nt spacing between sequential oligos. These lncRNA‐derived sequences (gray) were appended with unique barcodes and universal primer binding sites, resulting in a pool of 11,969 oligos of 153 bp ( Table EV1 ). The vertical lines in the lncRNA denote splice junctions. Second from left : schematic summarizing the design of each oligonucleotide. Second from right : reporter design. The oligonucleotide pool was cloned into a reporter plasmid as fusion transcript 3′ of fsSox2 (minCMV, minimal CMV promoter; pA, polyadenylation sequence). Far right : MPRA workflow. The fsSox2 ˜oligo reporter pool was transiently transfected into HeLa cells. Following 48 h of expression, cells were harvested and fractionated to isolate nuclei, and the nuclear enrichment of each oligo was quantified by targeted RNA sequencing. Matched whole‐cell lysates from unfractionated cells served as controls. Read mapping and normalization. A perfect match between the first 10 nt of the read and the barcode sequence was used to “map” the read. To guarantee robustness of the mapping procedure, we allowed for no more than two mismatches within the 90 basepairs upstream of the barcode (see “mapReads” function in our analysis package—please refer to the Code availability section). Counts were normalized for library size using the “normCounts” table (see analysis package and GEO data—please refer to the Code and Data availability sections). Counts for each nucleotide were modeled based on the normalized counts for each oligo. When nucleotide “A” overlapped with oligos i 1 , i 2 , i 3 , and i 4 , counts for this nucleotide were modeled by the median of counts for each of the individual oligos (i 1 –i 4 ; see “modelNucCounts” function in our analysis pipeline). The nucleotide counts were then used to infer differential regions by (1): finding candidate regions and assigning a summary statistic to each one of them and next (2): generating null candidates by permuting sample labels and using them to assign an empirical P ‐value to our candidate regions from step 1 to identify significant regions. Differential region‐calling correctly identifies nuclear retention elements in MALAT1 . Solid lines: per‐nucleotide abundances in the nuclear (red) and whole‐cell (gray) fractions, modeled for each nucleotide position along the MALAT1 transcript, based on the aggregate behavior of all oligos containing that nucleotide (shaded regions: ±SD, medians of six biological replicates).
Figure Legend Snippet: A Massively Parallel RNA Assay ( MPRNA ) to identify RNA nuclear enrichment signals Experimental overview. Far left : oligonucleotide pool design. Double‐stranded DNA (dsDNA) oligonucleotides were designed by computationally scanning 38 parental lncRNA transcripts ( Table EV1 ) in 110‐nt windows, with 10‐nt spacing between sequential oligos. These lncRNA‐derived sequences (gray) were appended with unique barcodes and universal primer binding sites, resulting in a pool of 11,969 oligos of 153 bp ( Table EV1 ). The vertical lines in the lncRNA denote splice junctions. Second from left : schematic summarizing the design of each oligonucleotide. Second from right : reporter design. The oligonucleotide pool was cloned into a reporter plasmid as fusion transcript 3′ of fsSox2 (minCMV, minimal CMV promoter; pA, polyadenylation sequence). Far right : MPRA workflow. The fsSox2 ˜oligo reporter pool was transiently transfected into HeLa cells. Following 48 h of expression, cells were harvested and fractionated to isolate nuclei, and the nuclear enrichment of each oligo was quantified by targeted RNA sequencing. Matched whole‐cell lysates from unfractionated cells served as controls. Read mapping and normalization. A perfect match between the first 10 nt of the read and the barcode sequence was used to “map” the read. To guarantee robustness of the mapping procedure, we allowed for no more than two mismatches within the 90 basepairs upstream of the barcode (see “mapReads” function in our analysis package—please refer to the Code availability section). Counts were normalized for library size using the “normCounts” table (see analysis package and GEO data—please refer to the Code and Data availability sections). Counts for each nucleotide were modeled based on the normalized counts for each oligo. When nucleotide “A” overlapped with oligos i 1 , i 2 , i 3 , and i 4 , counts for this nucleotide were modeled by the median of counts for each of the individual oligos (i 1 –i 4 ; see “modelNucCounts” function in our analysis pipeline). The nucleotide counts were then used to infer differential regions by (1): finding candidate regions and assigning a summary statistic to each one of them and next (2): generating null candidates by permuting sample labels and using them to assign an empirical P ‐value to our candidate regions from step 1 to identify significant regions. Differential region‐calling correctly identifies nuclear retention elements in MALAT1 . Solid lines: per‐nucleotide abundances in the nuclear (red) and whole‐cell (gray) fractions, modeled for each nucleotide position along the MALAT1 transcript, based on the aggregate behavior of all oligos containing that nucleotide (shaded regions: ±SD, medians of six biological replicates).

Techniques Used: Derivative Assay, Binding Assay, Clone Assay, Plasmid Preparation, Sequencing, Transfection, Expressing, RNA Sequencing Assay

Quality assessment of every MPRNA step The distribution of oligos in the cloned plasmid pool. (i) the single peak showing uniform counts for several different oligos indicates very little jackpotting, and (ii) entire oligo representation (small bump at zero counts). Nuclear enrichment of NEAT1 , GAPDH, and SNHG5 as determined by qRT–PCR in each biological replicate (left) (error bars: ±SD). Nuclear enrichment of median ± SD across the six replicates. Transfection efficiency of HeLa cells co‐transfected with a GFP plasmid using the protocol outlined in Materials and Methods . A recovery of > 70% of our initial oligo‐pool was obtained in each sample. On average, only 0.2% of the oligos (i.e., ˜25 oligos) was not detected in the nuclear fraction samples, and ˜0.4% (i.e., ˜50 oligos) in the Total (whole‐cell lysate) samples. Bar plots showing the mapping percentage for all reads of different samples from nuclear (N) and total fractions (T), separated for technical replicates (TR) and biological replicates (BR). Boxplots showing inter‐replicate differences between counts of the same oligo. Low technical variance was detected as indicated by low differences in counts among technical replicates. The solid horizontal line is the median while the lower and upper hinges correspond to the first and third quartiles (the 25 th and 75 th percentiles). The upper whisker extends from the hinge to the largest value no further than 1.5 × IQR from the hinge. The lower whisker extends from the hinge to the smallest value at most 1.5 × IQR of the hinge. Data beyond the end of the whiskers are outliers and are plotted individually. CDF plot of the nucleotides overlapping human RRD, mouse RRD, and other nucleotides in the human and mouse FIRRE loci. Similar to the MALAT1 Region M and Region E, the MPRNA recapitulated the function of the known RNA nuclear retention element RRD of the FIRRE locus. Since the experiments were performed in human HeLa cells, human FIRRE RRD was nuclear enriched, while the mouse FIRRE RRD did not influence nuclear enrichment of fsSox2 ( P ‐value: Mann–Whitney test comparison between RRD nucleotides of the human FIRRE transcript versus other non RRD nucleotides in the human and mouse FIRRE transcripts). Differential region‐calling correctly identified nuclear retention elements in FIRRE . Solid lines: per‐nucleotide abundances in the nuclear (red) and whole‐cell (gray) fractions, modeled for each position along the FIRRE transcript, based on the aggregate behavior of all oligos containing that nucleotide (shaded regions ±SD, medians for six biological replicates).
Figure Legend Snippet: Quality assessment of every MPRNA step The distribution of oligos in the cloned plasmid pool. (i) the single peak showing uniform counts for several different oligos indicates very little jackpotting, and (ii) entire oligo representation (small bump at zero counts). Nuclear enrichment of NEAT1 , GAPDH, and SNHG5 as determined by qRT–PCR in each biological replicate (left) (error bars: ±SD). Nuclear enrichment of median ± SD across the six replicates. Transfection efficiency of HeLa cells co‐transfected with a GFP plasmid using the protocol outlined in Materials and Methods . A recovery of > 70% of our initial oligo‐pool was obtained in each sample. On average, only 0.2% of the oligos (i.e., ˜25 oligos) was not detected in the nuclear fraction samples, and ˜0.4% (i.e., ˜50 oligos) in the Total (whole‐cell lysate) samples. Bar plots showing the mapping percentage for all reads of different samples from nuclear (N) and total fractions (T), separated for technical replicates (TR) and biological replicates (BR). Boxplots showing inter‐replicate differences between counts of the same oligo. Low technical variance was detected as indicated by low differences in counts among technical replicates. The solid horizontal line is the median while the lower and upper hinges correspond to the first and third quartiles (the 25 th and 75 th percentiles). The upper whisker extends from the hinge to the largest value no further than 1.5 × IQR from the hinge. The lower whisker extends from the hinge to the smallest value at most 1.5 × IQR of the hinge. Data beyond the end of the whiskers are outliers and are plotted individually. CDF plot of the nucleotides overlapping human RRD, mouse RRD, and other nucleotides in the human and mouse FIRRE loci. Similar to the MALAT1 Region M and Region E, the MPRNA recapitulated the function of the known RNA nuclear retention element RRD of the FIRRE locus. Since the experiments were performed in human HeLa cells, human FIRRE RRD was nuclear enriched, while the mouse FIRRE RRD did not influence nuclear enrichment of fsSox2 ( P ‐value: Mann–Whitney test comparison between RRD nucleotides of the human FIRRE transcript versus other non RRD nucleotides in the human and mouse FIRRE transcripts). Differential region‐calling correctly identified nuclear retention elements in FIRRE . Solid lines: per‐nucleotide abundances in the nuclear (red) and whole‐cell (gray) fractions, modeled for each position along the FIRRE transcript, based on the aggregate behavior of all oligos containing that nucleotide (shaded regions ±SD, medians for six biological replicates).

Techniques Used: Clone Assay, Plasmid Preparation, Quantitative RT-PCR, Transfection, Whisker Assay, MANN-WHITNEY

14) Product Images from "High-Throughput Sequencing of RNA Isolated by Cross- Linking and Immunoprecipitation (HITS-CLIP) to Determine Sites of Binding of CstF-64 on Nascent RNAs"

Article Title: High-Throughput Sequencing of RNA Isolated by Cross- Linking and Immunoprecipitation (HITS-CLIP) to Determine Sites of Binding of CstF-64 on Nascent RNAs

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

doi: 10.1007/978-1-62703-971-0_17

Purification of RNA oligomers and DNA amplimers after the CLIP procedure. ( a ) Autoradiograph of the 32 P-labeled RNA oligonucleotides with various sizes isolated as described in the text and subsequently ligated to the RA3 and RA5 adapters using the Illumina’s
Figure Legend Snippet: Purification of RNA oligomers and DNA amplimers after the CLIP procedure. ( a ) Autoradiograph of the 32 P-labeled RNA oligonucleotides with various sizes isolated as described in the text and subsequently ligated to the RA3 and RA5 adapters using the Illumina’s

Techniques Used: Purification, Cross-linking Immunoprecipitation, Autoradiography, Labeling, Isolation

15) Product Images from "High-throughput biochemical profiling reveals sequence determinants of dCas9 off-target binding and unbinding"

Article Title: High-throughput biochemical profiling reveals sequence determinants of dCas9 off-target binding and unbinding

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

doi: 10.1073/pnas.1700557114

Quantifying dCas9 binding behavior on a massively parallel array. ( A ) Experimental procedure for high-throughput biochemical profiling. A fluorescent DNA oligo hybridized to the dCas9 sgRNA was loaded into the apo-dCas9. In parallel, an Illumina sequencing-compatible DNA construct was both labeled and made double-stranded by extending a second fluorescent oligo. dCas9 was flowed into the chamber, allowing association with double-stranded DNA. A dissociation experiment was then performed by quantifying the decrease in dCas9 signal upon dilution or chase. ( B ) Example images taken in two channels on the array, Alexa Fluor 647-labeled DNA (red) and Cy3-labeled dCas9 (green). A 12-h incubation, meant to saturate the clusters with dCas9, separates association from dissociation experiments (dotted line). For most clusters, signal accumulated in the on-rate experiment largely remains throughout the dissociation. (Magnification: right nine panels, 16×.) ( C and D ) Examples of ( C ) association and ( D ) dissociation lines fit to different targets. The +1 base refers to the first base of the PAM, −1 to the most PAM-proximal base, and −20 to the most PAM-distal base. ( E ) The total number ( y axis) and percentage (in text) of possible targets profiled for each number of substitutions from the on-target site. Only a fraction of sequences with quantified on-rates are profiled for off-rates (blue) with high confidence. ( F ) Clusters per variant for targets with the given number of substitutions. AU, arbitrary units.
Figure Legend Snippet: Quantifying dCas9 binding behavior on a massively parallel array. ( A ) Experimental procedure for high-throughput biochemical profiling. A fluorescent DNA oligo hybridized to the dCas9 sgRNA was loaded into the apo-dCas9. In parallel, an Illumina sequencing-compatible DNA construct was both labeled and made double-stranded by extending a second fluorescent oligo. dCas9 was flowed into the chamber, allowing association with double-stranded DNA. A dissociation experiment was then performed by quantifying the decrease in dCas9 signal upon dilution or chase. ( B ) Example images taken in two channels on the array, Alexa Fluor 647-labeled DNA (red) and Cy3-labeled dCas9 (green). A 12-h incubation, meant to saturate the clusters with dCas9, separates association from dissociation experiments (dotted line). For most clusters, signal accumulated in the on-rate experiment largely remains throughout the dissociation. (Magnification: right nine panels, 16×.) ( C and D ) Examples of ( C ) association and ( D ) dissociation lines fit to different targets. The +1 base refers to the first base of the PAM, −1 to the most PAM-proximal base, and −20 to the most PAM-distal base. ( E ) The total number ( y axis) and percentage (in text) of possible targets profiled for each number of substitutions from the on-target site. Only a fraction of sequences with quantified on-rates are profiled for off-rates (blue) with high confidence. ( F ) Clusters per variant for targets with the given number of substitutions. AU, arbitrary units.

Techniques Used: Binding Assay, High Throughput Screening Assay, Sequencing, Construct, Labeling, Incubation, Variant Assay

16) Product Images from "High-throughput biochemical profiling reveals sequence determinants of dCas9 off-target binding and unbinding"

Article Title: High-throughput biochemical profiling reveals sequence determinants of dCas9 off-target binding and unbinding

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

doi: 10.1073/pnas.1700557114

Quantifying dCas9 binding behavior on a massively parallel array. ( A ) Experimental procedure for high-throughput biochemical profiling. A fluorescent DNA oligo hybridized to the dCas9 sgRNA was loaded into the apo-dCas9. In parallel, an Illumina sequencing-compatible DNA construct was both labeled and made double-stranded by extending a second fluorescent oligo. dCas9 was flowed into the chamber, allowing association with double-stranded DNA. A dissociation experiment was then performed by quantifying the decrease in dCas9 signal upon dilution or chase. ( B ) Example images taken in two channels on the array, Alexa Fluor 647-labeled DNA (red) and Cy3-labeled dCas9 (green). A 12-h incubation, meant to saturate the clusters with dCas9, separates association from dissociation experiments (dotted line). For most clusters, signal accumulated in the on-rate experiment largely remains throughout the dissociation. (Magnification: right nine panels, 16×.) ( C and D ) Examples of ( C ) association and ( D ) dissociation lines fit to different targets. The +1 base refers to the first base of the PAM, −1 to the most PAM-proximal base, and −20 to the most PAM-distal base. ( E ) The total number ( y axis) and percentage (in text) of possible targets profiled for each number of substitutions from the on-target site. Only a fraction of sequences with quantified on-rates are profiled for off-rates (blue) with high confidence. ( F ) Clusters per variant for targets with the given number of substitutions. AU, arbitrary units.
Figure Legend Snippet: Quantifying dCas9 binding behavior on a massively parallel array. ( A ) Experimental procedure for high-throughput biochemical profiling. A fluorescent DNA oligo hybridized to the dCas9 sgRNA was loaded into the apo-dCas9. In parallel, an Illumina sequencing-compatible DNA construct was both labeled and made double-stranded by extending a second fluorescent oligo. dCas9 was flowed into the chamber, allowing association with double-stranded DNA. A dissociation experiment was then performed by quantifying the decrease in dCas9 signal upon dilution or chase. ( B ) Example images taken in two channels on the array, Alexa Fluor 647-labeled DNA (red) and Cy3-labeled dCas9 (green). A 12-h incubation, meant to saturate the clusters with dCas9, separates association from dissociation experiments (dotted line). For most clusters, signal accumulated in the on-rate experiment largely remains throughout the dissociation. (Magnification: right nine panels, 16×.) ( C and D ) Examples of ( C ) association and ( D ) dissociation lines fit to different targets. The +1 base refers to the first base of the PAM, −1 to the most PAM-proximal base, and −20 to the most PAM-distal base. ( E ) The total number ( y axis) and percentage (in text) of possible targets profiled for each number of substitutions from the on-target site. Only a fraction of sequences with quantified on-rates are profiled for off-rates (blue) with high confidence. ( F ) Clusters per variant for targets with the given number of substitutions. AU, arbitrary units.

Techniques Used: Binding Assay, High Throughput Screening Assay, Sequencing, Construct, Labeling, Incubation, Variant Assay

17) Product Images from "Highly selective retrieval of accurate DNA utilizing a pool of in situ-replicated DNA from multiple next-generation sequencing platforms"

Article Title: Highly selective retrieval of accurate DNA utilizing a pool of in situ-replicated DNA from multiple next-generation sequencing platforms

Journal: Nucleic Acids Research

doi: 10.1093/nar/gky016

Schematic of in situ replication of DNA molecules from next-generation sequencing (NGS) platforms and subsequent PCR-based retrieval of target sequences. ( A ) Process flow chart for PCR-based methods for the retrieval of error-free DNA targets from an NGS-replica pool. ( B ) Preparation strategy of 454 GS Junior sequencing-based retrieval. Combinatorial barcode-tagged (CBT) pools were processed from microarray-synthesized oligonucleotides and subsequently ligated to the sheared genomic DNA as flanking sequences. The library was replicated in a sealed NGS plate. ( C ) Preparation strategy of a pre-NGS pool (MiSeq and Ion Proton). The barcoded library (cgc50 pool) was directly synthesized on a microarray. ( D ) Schematic of library replication in a MiSeq flow cell. ( E ) Schematic of library replication using melt-off DNA in the Ion Proton system. This process could be automatically performed using an Ion OneTouch™ ES system.
Figure Legend Snippet: Schematic of in situ replication of DNA molecules from next-generation sequencing (NGS) platforms and subsequent PCR-based retrieval of target sequences. ( A ) Process flow chart for PCR-based methods for the retrieval of error-free DNA targets from an NGS-replica pool. ( B ) Preparation strategy of 454 GS Junior sequencing-based retrieval. Combinatorial barcode-tagged (CBT) pools were processed from microarray-synthesized oligonucleotides and subsequently ligated to the sheared genomic DNA as flanking sequences. The library was replicated in a sealed NGS plate. ( C ) Preparation strategy of a pre-NGS pool (MiSeq and Ion Proton). The barcoded library (cgc50 pool) was directly synthesized on a microarray. ( D ) Schematic of library replication in a MiSeq flow cell. ( E ) Schematic of library replication using melt-off DNA in the Ion Proton system. This process could be automatically performed using an Ion OneTouch™ ES system.

Techniques Used: In Situ, Next-Generation Sequencing, Polymerase Chain Reaction, Flow Cytometry, Sequencing, Microarray, Synthesized

18) Product Images from "A transcriptome map of perennial ryegrass (Lolium perenne L.)"

Article Title: A transcriptome map of perennial ryegrass (Lolium perenne L.)

Journal: BMC Genomics

doi: 10.1186/1471-2164-13-140

Examples of SNP graphs observed in Lolium oligo pool assay (LOPA1) GoldenGate genotyping. SNP graphs are illustrated using the Software Illumina® GenomeStudio, version 2009.2. The normalized R (y-axis) is the normalized sum of intensities of the two dyes (Cy3 and Cy5), the normalized Theta (x-axis) is the deviation of Cy3 and Cy5 fluorescence from pure Cy3 and pure Cy5 signal (0 and 1). A normalized Theta value close to 0 and 1 is homozygous for SNP variant 1 and 2, respectively, a heterozygous sample is in between. The red, blue and purple ovals have the diameter of two standard deviations computed from the dispersal of the red, blue and purple dots, respectively. The numbers of plants in each cluster are indicated below the x-axis. ( A ) The 192 samples genotyped for SNP marker PTA.1021.C1 revealed fluorescence signal intensities close to 0, indicating assay failure. ( B ) Although the clustering algorithm at SNP PTA.1.C3 distinguished the three clusters at a GenTrain score of 0.40, such a genotyping pattern was considered inaccurate and this SNP was discarded from further analysis. ( C ) This illustration shows the SNP graph of monomorphic P9G02. ( D ) and ( E ) illustrate dominant SNPs being homozygous in one and heterozygous in the other mapping parent. For genetic linkage mapping, the markers PTA.109.C1 and PTA.291.C1 followed the segregation type nnxnp and lmxll, respectively [ 53 ]. Dots corresponding to the parents of the VrnA mapping population (which are represented in duplicates) are highlighted in yellow. Graph ( F ) shows a classical example of a SNP marker being heterozygous in both parents following the segregation pattern hkxhk.
Figure Legend Snippet: Examples of SNP graphs observed in Lolium oligo pool assay (LOPA1) GoldenGate genotyping. SNP graphs are illustrated using the Software Illumina® GenomeStudio, version 2009.2. The normalized R (y-axis) is the normalized sum of intensities of the two dyes (Cy3 and Cy5), the normalized Theta (x-axis) is the deviation of Cy3 and Cy5 fluorescence from pure Cy3 and pure Cy5 signal (0 and 1). A normalized Theta value close to 0 and 1 is homozygous for SNP variant 1 and 2, respectively, a heterozygous sample is in between. The red, blue and purple ovals have the diameter of two standard deviations computed from the dispersal of the red, blue and purple dots, respectively. The numbers of plants in each cluster are indicated below the x-axis. ( A ) The 192 samples genotyped for SNP marker PTA.1021.C1 revealed fluorescence signal intensities close to 0, indicating assay failure. ( B ) Although the clustering algorithm at SNP PTA.1.C3 distinguished the three clusters at a GenTrain score of 0.40, such a genotyping pattern was considered inaccurate and this SNP was discarded from further analysis. ( C ) This illustration shows the SNP graph of monomorphic P9G02. ( D ) and ( E ) illustrate dominant SNPs being homozygous in one and heterozygous in the other mapping parent. For genetic linkage mapping, the markers PTA.109.C1 and PTA.291.C1 followed the segregation type nnxnp and lmxll, respectively [ 53 ]. Dots corresponding to the parents of the VrnA mapping population (which are represented in duplicates) are highlighted in yellow. Graph ( F ) shows a classical example of a SNP marker being heterozygous in both parents following the segregation pattern hkxhk.

Techniques Used: Pool Assay, Software, Fluorescence, Variant Assay, Marker

19) Product Images from "High‐throughput identification of RNA nuclear enrichment sequences"

Article Title: High‐throughput identification of RNA nuclear enrichment sequences

Journal: The EMBO Journal

doi: 10.15252/embj.201798452

A Massively Parallel RNA Assay ( MPRNA ) to identify RNA nuclear enrichment signals Experimental overview. Far left : oligonucleotide pool design. Double‐stranded DNA (dsDNA) oligonucleotides were designed by computationally scanning 38 parental lncRNA transcripts ( Table EV1 ) in 110‐nt windows, with 10‐nt spacing between sequential oligos. These lncRNA‐derived sequences (gray) were appended with unique barcodes and universal primer binding sites, resulting in a pool of 11,969 oligos of 153 bp ( Table EV1 ). The vertical lines in the lncRNA denote splice junctions. Second from left : schematic summarizing the design of each oligonucleotide. Second from right : reporter design. The oligonucleotide pool was cloned into a reporter plasmid as fusion transcript 3′ of fsSox2 (minCMV, minimal CMV promoter; pA, polyadenylation sequence). Far right : MPRA workflow. The fsSox2 ˜oligo reporter pool was transiently transfected into HeLa cells. Following 48 h of expression, cells were harvested and fractionated to isolate nuclei, and the nuclear enrichment of each oligo was quantified by targeted RNA sequencing. Matched whole‐cell lysates from unfractionated cells served as controls. Read mapping and normalization. A perfect match between the first 10 nt of the read and the barcode sequence was used to “map” the read. To guarantee robustness of the mapping procedure, we allowed for no more than two mismatches within the 90 basepairs upstream of the barcode (see “mapReads” function in our analysis package—please refer to the Code availability section). Counts were normalized for library size using the “normCounts” table (see analysis package and GEO data—please refer to the Code and Data availability sections). Counts for each nucleotide were modeled based on the normalized counts for each oligo. When nucleotide “A” overlapped with oligos i 1 , i 2 , i 3 , and i 4 , counts for this nucleotide were modeled by the median of counts for each of the individual oligos (i 1 –i 4 ; see “modelNucCounts” function in our analysis pipeline). The nucleotide counts were then used to infer differential regions by (1): finding candidate regions and assigning a summary statistic to each one of them and next (2): generating null candidates by permuting sample labels and using them to assign an empirical P ‐value to our candidate regions from step 1 to identify significant regions. Differential region‐calling correctly identifies nuclear retention elements in MALAT1 . Solid lines: per‐nucleotide abundances in the nuclear (red) and whole‐cell (gray) fractions, modeled for each nucleotide position along the MALAT1 transcript, based on the aggregate behavior of all oligos containing that nucleotide (shaded regions: ±SD, medians of six biological replicates).
Figure Legend Snippet: A Massively Parallel RNA Assay ( MPRNA ) to identify RNA nuclear enrichment signals Experimental overview. Far left : oligonucleotide pool design. Double‐stranded DNA (dsDNA) oligonucleotides were designed by computationally scanning 38 parental lncRNA transcripts ( Table EV1 ) in 110‐nt windows, with 10‐nt spacing between sequential oligos. These lncRNA‐derived sequences (gray) were appended with unique barcodes and universal primer binding sites, resulting in a pool of 11,969 oligos of 153 bp ( Table EV1 ). The vertical lines in the lncRNA denote splice junctions. Second from left : schematic summarizing the design of each oligonucleotide. Second from right : reporter design. The oligonucleotide pool was cloned into a reporter plasmid as fusion transcript 3′ of fsSox2 (minCMV, minimal CMV promoter; pA, polyadenylation sequence). Far right : MPRA workflow. The fsSox2 ˜oligo reporter pool was transiently transfected into HeLa cells. Following 48 h of expression, cells were harvested and fractionated to isolate nuclei, and the nuclear enrichment of each oligo was quantified by targeted RNA sequencing. Matched whole‐cell lysates from unfractionated cells served as controls. Read mapping and normalization. A perfect match between the first 10 nt of the read and the barcode sequence was used to “map” the read. To guarantee robustness of the mapping procedure, we allowed for no more than two mismatches within the 90 basepairs upstream of the barcode (see “mapReads” function in our analysis package—please refer to the Code availability section). Counts were normalized for library size using the “normCounts” table (see analysis package and GEO data—please refer to the Code and Data availability sections). Counts for each nucleotide were modeled based on the normalized counts for each oligo. When nucleotide “A” overlapped with oligos i 1 , i 2 , i 3 , and i 4 , counts for this nucleotide were modeled by the median of counts for each of the individual oligos (i 1 –i 4 ; see “modelNucCounts” function in our analysis pipeline). The nucleotide counts were then used to infer differential regions by (1): finding candidate regions and assigning a summary statistic to each one of them and next (2): generating null candidates by permuting sample labels and using them to assign an empirical P ‐value to our candidate regions from step 1 to identify significant regions. Differential region‐calling correctly identifies nuclear retention elements in MALAT1 . Solid lines: per‐nucleotide abundances in the nuclear (red) and whole‐cell (gray) fractions, modeled for each nucleotide position along the MALAT1 transcript, based on the aggregate behavior of all oligos containing that nucleotide (shaded regions: ±SD, medians of six biological replicates).

Techniques Used: Derivative Assay, Binding Assay, Clone Assay, Plasmid Preparation, Sequencing, Transfection, Expressing, RNA Sequencing Assay

20) Product Images from "The landscape of mouse meiotic double-strand break formation, processing and repair"

Article Title: The landscape of mouse meiotic double-strand break formation, processing and repair

Journal: Cell

doi: 10.1016/j.cell.2016.09.035

Analysis of DSB Resection (A) General resection trends. Heat map shows locally normalized Crick-strand SSDS coverage (10-bp bins) relative to centers of SPO11-oligo hotspots that overlap SSDS hotspots and that have no other hotspot within 5 kb. Hotspots were ordered by distance from 1005 bp left of the center to the point right of the center where cumulative SSDS signal was 90% of total. (B) Combining SPO11-oligo and SSDS data to estimate the length distribution of resection tracts genome-wide. The ssDNA coverage at a given position is a function of the nearby DSB distribution and the per-DSB resection profile R . .
Figure Legend Snippet: Analysis of DSB Resection (A) General resection trends. Heat map shows locally normalized Crick-strand SSDS coverage (10-bp bins) relative to centers of SPO11-oligo hotspots that overlap SSDS hotspots and that have no other hotspot within 5 kb. Hotspots were ordered by distance from 1005 bp left of the center to the point right of the center where cumulative SSDS signal was 90% of total. (B) Combining SPO11-oligo and SSDS data to estimate the length distribution of resection tracts genome-wide. The ssDNA coverage at a given position is a function of the nearby DSB distribution and the per-DSB resection profile R . .

Techniques Used: Genome Wide

Spatial Relationships Between PRDM9 Binding, H3K4me3, and DSBs . (B) H3K4me3 is often highly asymmetric around hotspots. Heat maps (data in 5-bp bins) were ordered according to H3K4me3 asymmetry. Data were locally normalized, so color-coding reflects the local spatial pattern, not relative signal strength between hotspots. . (D) The hotspot-enriched 12-bp motif and its disposition within the larger PRDM9 B6 binding site. (E) Asymmetric average profile of SPO11 oligos (15-bp Hann filter) around motif midpoints (n=9060). (F) SPO11-oligo spatial classes from k-means clustering. (G) H3K4me3 patterns are similar despite different SPO11-oligo patterns (15-bp Hann filter) between the motif classes from panel F. legend. In panel I, a value of 1 was added to each hotspot to permit plotting of hotspots with no H3K4me3 tags. (J) Schematic of modular PRDM9 DNA binding and histone methylation activities. ZnF, zinc-finger domain; MTase, methyltransferase domain. (K) H3K4me3 is an imperfect predictor of DSB frequency. SPO11 oligos and H3K4me3 tag counts were summed in the 1001-bp around hotspot centers. One H3K4me3 tag was added to each hotspot to permit plotting of hotspots with no H3K4me3 tags. Eight outliers (H3K4me3 > 10 4 ) are not shown. .
Figure Legend Snippet: Spatial Relationships Between PRDM9 Binding, H3K4me3, and DSBs . (B) H3K4me3 is often highly asymmetric around hotspots. Heat maps (data in 5-bp bins) were ordered according to H3K4me3 asymmetry. Data were locally normalized, so color-coding reflects the local spatial pattern, not relative signal strength between hotspots. . (D) The hotspot-enriched 12-bp motif and its disposition within the larger PRDM9 B6 binding site. (E) Asymmetric average profile of SPO11 oligos (15-bp Hann filter) around motif midpoints (n=9060). (F) SPO11-oligo spatial classes from k-means clustering. (G) H3K4me3 patterns are similar despite different SPO11-oligo patterns (15-bp Hann filter) between the motif classes from panel F. legend. In panel I, a value of 1 was added to each hotspot to permit plotting of hotspots with no H3K4me3 tags. (J) Schematic of modular PRDM9 DNA binding and histone methylation activities. ZnF, zinc-finger domain; MTase, methyltransferase domain. (K) H3K4me3 is an imperfect predictor of DSB frequency. SPO11 oligos and H3K4me3 tag counts were summed in the 1001-bp around hotspot centers. One H3K4me3 tag was added to each hotspot to permit plotting of hotspots with no H3K4me3 tags. Eight outliers (H3K4me3 > 10 4 ) are not shown. .

Techniques Used: Binding Assay, Methylation

Large-Scale Patterns of DSB Formation and Recombination ). SPO11-oligo density was exceptionally low in the non-PAR segments of the sex chromosomes, but very high in the PAR. The crossover rate in the PAR was set at 50 cM. (C) The greater crossover density on smaller autosomes is explained only in part by the higher SPO11-oligo density. .
Figure Legend Snippet: Large-Scale Patterns of DSB Formation and Recombination ). SPO11-oligo density was exceptionally low in the non-PAR segments of the sex chromosomes, but very high in the PAR. The crossover rate in the PAR was set at 50 cM. (C) The greater crossover density on smaller autosomes is explained only in part by the higher SPO11-oligo density. .

Techniques Used:

Nucleotide-Resolution Map of Meiotic DSBs in Wild-Type Mice (A) Early steps in recombination and the protein–DNA complexes (SPO11 oligos and ssDNA bound by DMC1 and RAD51) used to generate genome-wide recombination initiation maps. (B) SPO11 oligos immunoprecipitated (IP) from B6 mouse spermatocytes, deproteinized, 3′-end-labeled, and resolved in a denaturing 15% polyacrylamide gel. Anti-SPO11 antibody was omitted from the mock IP processed in parallel. (C) Length distribution of SPO11 oligos that map uniquely or to multiple sites. Oligos appear longer on gels (panel B) because of nucleotides added for labeling and amino acid(s) left after SPO11 proteolysis. (D) SPO11-oligo map (smoothed with a 1001-bp Hann filter) compared to positions of four known crossover hotspots ( A1–A4 ). ) in a 3001-bp window around hotspot A3 . SSDS coverage at each position was normalized to the total strand-specific coverage in the genome and multiplied by 10 6 . (F) In SSDS hotspots (n=18,294), SPO11-oligo counts correlated strongly (Pearson's r ) with SSDS tag counts. One SPO11-oligo read was added to permit plotting of hotspots with no oligos. (G) Distribution of SPO11 oligos (51-bp Hann filter) and SSDS coverage around centers of SSDS hotspots. .
Figure Legend Snippet: Nucleotide-Resolution Map of Meiotic DSBs in Wild-Type Mice (A) Early steps in recombination and the protein–DNA complexes (SPO11 oligos and ssDNA bound by DMC1 and RAD51) used to generate genome-wide recombination initiation maps. (B) SPO11 oligos immunoprecipitated (IP) from B6 mouse spermatocytes, deproteinized, 3′-end-labeled, and resolved in a denaturing 15% polyacrylamide gel. Anti-SPO11 antibody was omitted from the mock IP processed in parallel. (C) Length distribution of SPO11 oligos that map uniquely or to multiple sites. Oligos appear longer on gels (panel B) because of nucleotides added for labeling and amino acid(s) left after SPO11 proteolysis. (D) SPO11-oligo map (smoothed with a 1001-bp Hann filter) compared to positions of four known crossover hotspots ( A1–A4 ). ) in a 3001-bp window around hotspot A3 . SSDS coverage at each position was normalized to the total strand-specific coverage in the genome and multiplied by 10 6 . (F) In SSDS hotspots (n=18,294), SPO11-oligo counts correlated strongly (Pearson's r ) with SSDS tag counts. One SPO11-oligo read was added to permit plotting of hotspots with no oligos. (G) Distribution of SPO11 oligos (51-bp Hann filter) and SSDS coverage around centers of SSDS hotspots. .

Techniques Used: Mouse Assay, Genome Wide, Immunoprecipitation, Labeling

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

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

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

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

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

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

22) Product Images from "Investigation of Experimental Factors That Underlie BRCA1/2 mRNA Isoform Expression Variation: Recommendations for Utilizing Targeted RNA Sequencing to Evaluate Potential Spliceogenic Variants"

Article Title: Investigation of Experimental Factors That Underlie BRCA1/2 mRNA Isoform Expression Variation: Recommendations for Utilizing Targeted RNA Sequencing to Evaluate Potential Spliceogenic Variants

Journal: Frontiers in Oncology

doi: 10.3389/fonc.2018.00140

Relative expression of BRCA1 and BRCA2 mRNA isoforms in rare variant samples compared to controls. (A) Natural expression ranges of mRNA splice isoforms calculated from lymphoblastoid cell lines (LCLs) not containing any known spliceogenic variants in BRCA1 (A) and BRCA2 (C) . Colored symbols overlaid indicate the relative mRNA isoform expression in LCLs containing known BRCA1 (B) or BRCA2 (D) splice disrupting variants. Only mRNA splice isoforms that were detected by more than 10 reads in at least two controls were included. Mean and upper and lower limits shown for each isoform [SE (95%)].
Figure Legend Snippet: Relative expression of BRCA1 and BRCA2 mRNA isoforms in rare variant samples compared to controls. (A) Natural expression ranges of mRNA splice isoforms calculated from lymphoblastoid cell lines (LCLs) not containing any known spliceogenic variants in BRCA1 (A) and BRCA2 (C) . Colored symbols overlaid indicate the relative mRNA isoform expression in LCLs containing known BRCA1 (B) or BRCA2 (D) splice disrupting variants. Only mRNA splice isoforms that were detected by more than 10 reads in at least two controls were included. Mean and upper and lower limits shown for each isoform [SE (95%)].

Techniques Used: Expressing, Variant Assay

BRCA2 mRNA isoforms detected at six time points in an lymphoblastoid cell line (sample #7, Table S1 in Supplementary Material) treated with an nonsense-mediated decay inhibitor. A freeze–thaw process was undertaken after time points two and four. Three technical replicates are listed under each time point.
Figure Legend Snippet: BRCA2 mRNA isoforms detected at six time points in an lymphoblastoid cell line (sample #7, Table S1 in Supplementary Material) treated with an nonsense-mediated decay inhibitor. A freeze–thaw process was undertaken after time points two and four. Three technical replicates are listed under each time point.

Techniques Used:

23) Product Images from "A low-cost open-source SNP genotyping platform for association mapping applications"

Article Title: A low-cost open-source SNP genotyping platform for association mapping applications

Journal: Genome Biology

doi: 10.1186/gb-2005-6-12-r105

Principle of OLA-based SNP genotyping. (a) For each polymorphism, a set of three genotyping oligos are allowed to anneal to denatured PCR product (blue) in the presence of Taq DNA ligase. Ligation of up- and downstream oligos occurs only if there is a perfect match to template. Upstream oligos are color-coded gray (M13 forward amplification primer sequence), red/green (a pair of barcode sequences), and black (assay-specific sequence flanking the query SNP). The downstream oligo is 5'-phosphorylated, and color-coded gray (reverse complemented sequence of the M13 reverse amplification primer), and black (assay-specific flanking sequence). (b) Addition of common M13 primers (gray) allows amplification of all ligated products. (c) After arraying amplified OLA products, membranes are hybridized with probes complementary to the barcode sequences. Probes can be fluorescently labeled with infrared (IR) fluors and both alleles hybridized simultaneously, or radiolabeled and hybridized sequentially.
Figure Legend Snippet: Principle of OLA-based SNP genotyping. (a) For each polymorphism, a set of three genotyping oligos are allowed to anneal to denatured PCR product (blue) in the presence of Taq DNA ligase. Ligation of up- and downstream oligos occurs only if there is a perfect match to template. Upstream oligos are color-coded gray (M13 forward amplification primer sequence), red/green (a pair of barcode sequences), and black (assay-specific sequence flanking the query SNP). The downstream oligo is 5'-phosphorylated, and color-coded gray (reverse complemented sequence of the M13 reverse amplification primer), and black (assay-specific flanking sequence). (b) Addition of common M13 primers (gray) allows amplification of all ligated products. (c) After arraying amplified OLA products, membranes are hybridized with probes complementary to the barcode sequences. Probes can be fluorescently labeled with infrared (IR) fluors and both alleles hybridized simultaneously, or radiolabeled and hybridized sequentially.

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

24) Product Images from "Combining Different mRNA Capture Methods to Analyze the Transcriptome: Analysis of the Xenopus laevis Transcriptome"

Article Title: Combining Different mRNA Capture Methods to Analyze the Transcriptome: Analysis of the Xenopus laevis Transcriptome

Journal: PLoS ONE

doi: 10.1371/journal.pone.0077700

Comparison of de novo transcriptome assemblies. A. Transcripts assembled by the Abyss assembler from oligo-dT and Cap-captured mRNA libraries were aligned to the X. tropicalis ENSEMBL annotated transcripts using BlastX. The number of ENSEMBL transcripts that were matched by each assembler:library pair are represented as a Venn Diagram. The number of transcripts present in each assembler:library pair are listed in Table 2. Because multiple sequences from each de novo assembly align to X. tropicalis genes these numbers are omitted from the figure for the sake of simplicity. B. Same comparison as in A, except that Velvet was used as the assembler instead of Abyss. C. Transcripts assembled by Abyss or Velvet from Cap-capture mRNA libraries were aligned to the X. tropicalis ENSEMBL transcripts using BLAT. Unique and common ENSEMBL transcripts are represented by a Venn Diagram D. Same comparison as in C, except that dT libraries are compared instead of Cap-captured libraries. E-H. Fraction of each ENSEMBL transcript covered by transcripts assembled using indicated assembler:library pair (from A-D) was calculated.
Figure Legend Snippet: Comparison of de novo transcriptome assemblies. A. Transcripts assembled by the Abyss assembler from oligo-dT and Cap-captured mRNA libraries were aligned to the X. tropicalis ENSEMBL annotated transcripts using BlastX. The number of ENSEMBL transcripts that were matched by each assembler:library pair are represented as a Venn Diagram. The number of transcripts present in each assembler:library pair are listed in Table 2. Because multiple sequences from each de novo assembly align to X. tropicalis genes these numbers are omitted from the figure for the sake of simplicity. B. Same comparison as in A, except that Velvet was used as the assembler instead of Abyss. C. Transcripts assembled by Abyss or Velvet from Cap-capture mRNA libraries were aligned to the X. tropicalis ENSEMBL transcripts using BLAT. Unique and common ENSEMBL transcripts are represented by a Venn Diagram D. Same comparison as in C, except that dT libraries are compared instead of Cap-captured libraries. E-H. Fraction of each ENSEMBL transcript covered by transcripts assembled using indicated assembler:library pair (from A-D) was calculated.

Techniques Used:

Comparison or mRNA-seq libraries from oligo-dT and Cap-captured mRNA. A-B. Reads from dT or Cap-capture prepared libraries were aligned to sequences consisting of the 5’ or 3’ 25% of Refseq mRNAs. Reads aligning to the 5’ and 3’ portions of the transcript are plotted. Blue line indicates a ratio of one. C. Reads from mRNA-seq libraries prepared using oligo-dT captured mRNAs from mitotic and interphase Xenopus egg extract were aligned to the Xenopus laevis Unigene database. Relative abundance of each mRNA in mitotic and interphase extracts is plotted. Red points highlight two-fold differences between mitotic and interphase samples. D. Same experiment as in panel C except that the mRNAs were purified using Cap-capture prior to library preparation. Red points highlight two-fold differences. E. Scatterplot of mRNA abundance in oligo-dT-captured and Cap-captured mRNA libraries. F. The ratio of reads per mRNA in mitotic and interphase extracts are plotted for oligo-dT captured mRNAs (X-axis, data from panel A) and cap-captured mRNAs (Y-axis data from panel B). The colored points correspond to mRNAs with known changes in poly-A tail length (cdk1, green; Eg2/aurora-a, orange; mos, red; Xlcl1, yellow). Quadrants highlight two-fold differences between samples.
Figure Legend Snippet: Comparison or mRNA-seq libraries from oligo-dT and Cap-captured mRNA. A-B. Reads from dT or Cap-capture prepared libraries were aligned to sequences consisting of the 5’ or 3’ 25% of Refseq mRNAs. Reads aligning to the 5’ and 3’ portions of the transcript are plotted. Blue line indicates a ratio of one. C. Reads from mRNA-seq libraries prepared using oligo-dT captured mRNAs from mitotic and interphase Xenopus egg extract were aligned to the Xenopus laevis Unigene database. Relative abundance of each mRNA in mitotic and interphase extracts is plotted. Red points highlight two-fold differences between mitotic and interphase samples. D. Same experiment as in panel C except that the mRNAs were purified using Cap-capture prior to library preparation. Red points highlight two-fold differences. E. Scatterplot of mRNA abundance in oligo-dT-captured and Cap-captured mRNA libraries. F. The ratio of reads per mRNA in mitotic and interphase extracts are plotted for oligo-dT captured mRNAs (X-axis, data from panel A) and cap-captured mRNAs (Y-axis data from panel B). The colored points correspond to mRNAs with known changes in poly-A tail length (cdk1, green; Eg2/aurora-a, orange; mos, red; Xlcl1, yellow). Quadrants highlight two-fold differences between samples.

Techniques Used: Purification

Poly-A tail analysis of selected mRNAs. mRNAs that exhibited changes in Mitosis:Interphase (M:IF) abundance ratios in oligo-dT-captured samples, but not in Cap-captured samples were analyzed for poly-A tail length using the ePAT assay and anchored TVN reverse transcription controls. A. Six mRNAs with high M:IF ratios (aurora-a, esco2, fbox5, stx11, march7, and hexim1) showed longer poly-A tails in mitotic extract. Two mRNA (setd8 and MGC83922) with a low M:IF ratio showed very modest changes in poly-A tail lengths between Mitosis and Interphase. Red asterix indicates the position of the prominent TVN PCR product that is quantified in B. B. The amount of PCR product contained in the TVN-RT PCR reactions (red asterix in A) were quantified. The ratio of the amount of PCR product in Mitotic to Interphase extracts is presented in the first line. The ratio of each mRNA in Mitotic and Interphase extracts as determined by RNA-seq is presented below the PCR derived ratios for comparison. In addition five mRNAs with high M:IF ratios (aurora-1, fbox5, stx11, march7, and hexim1) had increased amounts of minimal poly-A tail PCR products in mitosis compared to interphase in TVN controls (quantified below gel) while both setd8 and MGC88922 had higher levels of TVN PCR products in interphase compared to mitotic extracts TVN PCR products indicate mRNAs with poly-A tails of 18 As. Line traces of ePAT PCR reactions presented in panel A. Black lines indicate traces from mitotic extract and red lines indicate traces from interaphse extract. D. Semi-quantitative PCR for each of the mRNAs tested in A was performed on RNA from mitotic and interphase extracts. Random hexamers were used to prime reverse transcription for these reactions. A second experiment showing very similar results is present in Figure S2.
Figure Legend Snippet: Poly-A tail analysis of selected mRNAs. mRNAs that exhibited changes in Mitosis:Interphase (M:IF) abundance ratios in oligo-dT-captured samples, but not in Cap-captured samples were analyzed for poly-A tail length using the ePAT assay and anchored TVN reverse transcription controls. A. Six mRNAs with high M:IF ratios (aurora-a, esco2, fbox5, stx11, march7, and hexim1) showed longer poly-A tails in mitotic extract. Two mRNA (setd8 and MGC83922) with a low M:IF ratio showed very modest changes in poly-A tail lengths between Mitosis and Interphase. Red asterix indicates the position of the prominent TVN PCR product that is quantified in B. B. The amount of PCR product contained in the TVN-RT PCR reactions (red asterix in A) were quantified. The ratio of the amount of PCR product in Mitotic to Interphase extracts is presented in the first line. The ratio of each mRNA in Mitotic and Interphase extracts as determined by RNA-seq is presented below the PCR derived ratios for comparison. In addition five mRNAs with high M:IF ratios (aurora-1, fbox5, stx11, march7, and hexim1) had increased amounts of minimal poly-A tail PCR products in mitosis compared to interphase in TVN controls (quantified below gel) while both setd8 and MGC88922 had higher levels of TVN PCR products in interphase compared to mitotic extracts TVN PCR products indicate mRNAs with poly-A tails of 18 As. Line traces of ePAT PCR reactions presented in panel A. Black lines indicate traces from mitotic extract and red lines indicate traces from interaphse extract. D. Semi-quantitative PCR for each of the mRNAs tested in A was performed on RNA from mitotic and interphase extracts. Random hexamers were used to prime reverse transcription for these reactions. A second experiment showing very similar results is present in Figure S2.

Techniques Used: Polymerase Chain Reaction, Reverse Transcription Polymerase Chain Reaction, RNA Sequencing Assay, Derivative Assay, Real-time Polymerase Chain Reaction

25) Product Images from "Hierarchical regulation of the genome: global changes in nucleosome organization potentiate genome response"

Article Title: Hierarchical regulation of the genome: global changes in nucleosome organization potentiate genome response

Journal: Oncotarget

doi: 10.18632/oncotarget.6841

Mapping nucleosome distributions following KSHV reactivation using mTSS-seq technique and mTSS-seq validation A. Experimental design for mapping nucleosome distributions following KSHV reactivation using our newly developed mTSS-seq technique. B. Validation of mTSS-seq using quantitative PCR for both on-target regions of the genome (within the sequence-capture region, two-kb region centered on a TSS) and off-target regions of the genome (not within the sequence-capture region, outside the two-region centered on a TSS). Quantitative PCR was performed for on- and off-target regions of the genome for both RHOC and ITGA4. The y-axis shows C t values. On average, C t values between the on- and off-targets of the sequence-captured libraries differ by 10.5 cycles. C. The periodic occurrence of AA/TT/AT/TA dinucleotides was calculated for all nucleosomal-sized fragments (147-148 bp) at the 0 hour time point. The x-axis represents the distance from the dyad axis. The y-axis is the frequency of AA/TT/AT/TA dinucleotides. An A/T-containing dinucleotide periodicity is seen every 10 bp at the 0 hour time point. D. Alignment of the midpoint fragments (purple) from mTSS-seq to the human genome shown in the UCSC Genome Browser ( http://genome.ucsc.edu ). Zooming in 5000X on human chromosome 2 to a six-kb window with two-kb of midpoint fragments at 0 hour time point (purple lines), along with the sequence-capture oligos and previously-published human-nucleosome distribution map for cell line GM18508 (red lines, (Gaffney et al., 2012)) at the TSSs of PUS10 and PEX13. The x-axis is genomic location. The y-axis is scaled reads per million.
Figure Legend Snippet: Mapping nucleosome distributions following KSHV reactivation using mTSS-seq technique and mTSS-seq validation A. Experimental design for mapping nucleosome distributions following KSHV reactivation using our newly developed mTSS-seq technique. B. Validation of mTSS-seq using quantitative PCR for both on-target regions of the genome (within the sequence-capture region, two-kb region centered on a TSS) and off-target regions of the genome (not within the sequence-capture region, outside the two-region centered on a TSS). Quantitative PCR was performed for on- and off-target regions of the genome for both RHOC and ITGA4. The y-axis shows C t values. On average, C t values between the on- and off-targets of the sequence-captured libraries differ by 10.5 cycles. C. The periodic occurrence of AA/TT/AT/TA dinucleotides was calculated for all nucleosomal-sized fragments (147-148 bp) at the 0 hour time point. The x-axis represents the distance from the dyad axis. The y-axis is the frequency of AA/TT/AT/TA dinucleotides. An A/T-containing dinucleotide periodicity is seen every 10 bp at the 0 hour time point. D. Alignment of the midpoint fragments (purple) from mTSS-seq to the human genome shown in the UCSC Genome Browser ( http://genome.ucsc.edu ). Zooming in 5000X on human chromosome 2 to a six-kb window with two-kb of midpoint fragments at 0 hour time point (purple lines), along with the sequence-capture oligos and previously-published human-nucleosome distribution map for cell line GM18508 (red lines, (Gaffney et al., 2012)) at the TSSs of PUS10 and PEX13. The x-axis is genomic location. The y-axis is scaled reads per million.

Techniques Used: Real-time Polymerase Chain Reaction, Sequencing

26) Product Images from "Novel insights into RNP granules by employing the trypanosome's microtubule skeleton as a molecular sieve"

Article Title: Novel insights into RNP granules by employing the trypanosome's microtubule skeleton as a molecular sieve

Journal: Nucleic Acids Research

doi: 10.1093/nar/gkv731

Analysis of RNA samples taken during the granule enrichment. ( A ) Northern blots. Northern gels loaded with total RNA samples were probed for total mRNAs and for 18S rRNA. SL = spliced leader RNA. ( B ) The percentage of mRNA in the ‘granule’ fraction of non-starved and starved cells was quantified from northern blots of three independent experiments. Note that this percentage is calculated based on total mRNA being the sum of SN1, SN2, SN3, SN4 and P4; ‘T’ is mRNA prepared in parallel as a control for mRNA quality and is not equivalent to the other samples. ( C and D ) Starved trypanosomes were stained for total mRNA with a fluorescent oligo antisense to the mini-exon sequence (RNA FISH): one representative cell is shown in C. The fraction of fluorescence in granules in comparison to total fluorescence was quantified from Z-stack projections of 49 cells (all were in a early cell cycle stage prior to the division of their kinetoplast and nucleus). Non-starved trypanosomes had no visible granules (see Figures 8 ) and hybridization with an oligo sense to the spliced leader sequence (negative control) gave a 14-fold weaker total signal (data not shown). This experiment is one representative of several.
Figure Legend Snippet: Analysis of RNA samples taken during the granule enrichment. ( A ) Northern blots. Northern gels loaded with total RNA samples were probed for total mRNAs and for 18S rRNA. SL = spliced leader RNA. ( B ) The percentage of mRNA in the ‘granule’ fraction of non-starved and starved cells was quantified from northern blots of three independent experiments. Note that this percentage is calculated based on total mRNA being the sum of SN1, SN2, SN3, SN4 and P4; ‘T’ is mRNA prepared in parallel as a control for mRNA quality and is not equivalent to the other samples. ( C and D ) Starved trypanosomes were stained for total mRNA with a fluorescent oligo antisense to the mini-exon sequence (RNA FISH): one representative cell is shown in C. The fraction of fluorescence in granules in comparison to total fluorescence was quantified from Z-stack projections of 49 cells (all were in a early cell cycle stage prior to the division of their kinetoplast and nucleus). Non-starved trypanosomes had no visible granules (see Figures 8 ) and hybridization with an oligo sense to the spliced leader sequence (negative control) gave a 14-fold weaker total signal (data not shown). This experiment is one representative of several.

Techniques Used: Northern Blot, Staining, Sequencing, Fluorescence In Situ Hybridization, Fluorescence, Hybridization, Negative Control

27) Product Images from "TSS-EMOTE, a refined protocol for a more complete and less biased global mapping of transcription start sites in bacterial pathogens"

Article Title: TSS-EMOTE, a refined protocol for a more complete and less biased global mapping of transcription start sites in bacterial pathogens

Journal: BMC Genomics

doi: 10.1186/s12864-016-3211-3

TSS-EMOTE flowchart. The TSS-EMOTE assay consists of a wet-lab library preparation (panels a to g ) and in silico analyses (panel H to N). An asterisk continually marks the original 5’-base of tri-phosphorylated RNA (thin red line). a Total RNA is purified, and digested with XRN1 5’-exonuclease, which removes the vast majority of 5’ mono-phosphorylated RNA from the sample (including 16S and 23S rRNA). b and c The XRN1 treated RNA is mixed with large excess of a synthetic RNA oligo (Rp6, shown in blue), and split into two pools. Both pools receive T4 RNA ligase, but only the “+RppH” pool is co-treated with RppH, an enzyme that converts 5’ tri-phosphorylated ends to mono-phosphorylated ends, thus allowing the ligase to use them as substrates. d and e After the ligation reaction, a semi-random primer is used to reverse-transcribe the RNA and simultaneously add a 2.0 Illumina adapter (black “B”). This results in cDNA with a 2.0 Illumina adaptor (for reverse reads in paired-end sequencing) at the 5’-end and if the template RNA was ligated to an Rp6 oligo, then the cDNA will also have a complementary sequence to Rp6 at the 3’-end (cRp6). f PCR is used to specifically amplify cDNAs that carry the 2.0 Illumina adaptor and cRp6 sequences. This step moreover adds a 1.0 Illumina adaptor (for forward reads in paired-end sequencing) and a sample-specific 4-base EMOTE barcode (blue line and “XXX”, respectively) to index the molecules (different barcodes for the -RppH and + RppH pools). The barcode of the -RppH pool will designate molecules where the XRN1 treatments has been incomplete, and this information is incorporated into the in silico analysis (see below). g The barcoded DNA from various samples (and pools) can be mixed, and loaded directly into an Illumina HiSeq machine. Millions of 50 nt sequences are obtained, each of which will span the EMOTE barcode, both known and random sections of the Rp6 oligo (see Methods ), and it will reveal the first 20 nt of the native 5’-end of the ligated RNA molecule. These 20 nt are sufficient to map the vast majority of 5’-ends to a unique position on the small genomes of the bacteria in this study. However, longer Illumina reads (and thus longer mapping sequences) can be used if the TSSs are in repeated regions or if large-genome organisms, such as humans, are being examined. h The in silico pipeline input consists of stranded RNA-seq reads for one or multiple biological replicates in FASTQ format. Each replicate includes a FASTQ for the -RppH pool and another for the + RppH pool. i The FASTQ files go through EMOTE-conv software [ 51 ] that parses the reads, aligns them to the genome, and perform the quantification. Thus, for each genomic position we obtain the number of reads whose first nucleotide align at this genomic position, and on which strand it maps. The counts are further corrected for PCR biases by looking at the unique molecular identifiers (UMIs) sequences available in the unaligned part of the EMOTE read. j Quantification counts obtained for + RppH and -RppH pools are compared through a beta-binomial model that tests whether the identified 5’ ends in the + RppH pool is significantly enriched over the identified 5’ ends in the -RppH pool at a given position. The process results in a p-value that reflects our confidence in the genomic position to be enriched in the + RppH pool of the biological replicate. k The p-values of all the biological replicates are combined into a single p-value with Fisher’s method. l and m To correct the p-values for multiple testing across all genomic positions, the false discovery rate (FDR) is evaluated and only those with a FDR ≤ 0.01 are considered to be TSSs. Note also that for the FDR is only calculated for genomic positions with at least 5 detected ligation-events in at least one of the + RppH pools (UMI ≥ 5). n The TSSs then enter an annotation process that retrieve their surrounding sequence and downstream ORFs. TSSs separated by less than 5 bp are clustered together. Finally, to draw a global picture of operon structures, an independent detection of transcription terminators is operated with the software TransTermHP [ 39 ]. o Sequence of the RNA oligo Rp6 and a typical Illumina sequencing read from a TSS-EMOTE experiment. The Recognition Sequence serves as priming site for the PCR in panel F. UMI: The randomly incorporated nucleotides in the Rp6 oligo that serves to whether Illumina reads with identical Mapping Sequences originate from separate ligation events. CS: Control Sequence. EB: EMOTE barcode to index the Illumina reads. An asterisk indicates the 5’ nucleotide of the original RNA molecule
Figure Legend Snippet: TSS-EMOTE flowchart. The TSS-EMOTE assay consists of a wet-lab library preparation (panels a to g ) and in silico analyses (panel H to N). An asterisk continually marks the original 5’-base of tri-phosphorylated RNA (thin red line). a Total RNA is purified, and digested with XRN1 5’-exonuclease, which removes the vast majority of 5’ mono-phosphorylated RNA from the sample (including 16S and 23S rRNA). b and c The XRN1 treated RNA is mixed with large excess of a synthetic RNA oligo (Rp6, shown in blue), and split into two pools. Both pools receive T4 RNA ligase, but only the “+RppH” pool is co-treated with RppH, an enzyme that converts 5’ tri-phosphorylated ends to mono-phosphorylated ends, thus allowing the ligase to use them as substrates. d and e After the ligation reaction, a semi-random primer is used to reverse-transcribe the RNA and simultaneously add a 2.0 Illumina adapter (black “B”). This results in cDNA with a 2.0 Illumina adaptor (for reverse reads in paired-end sequencing) at the 5’-end and if the template RNA was ligated to an Rp6 oligo, then the cDNA will also have a complementary sequence to Rp6 at the 3’-end (cRp6). f PCR is used to specifically amplify cDNAs that carry the 2.0 Illumina adaptor and cRp6 sequences. This step moreover adds a 1.0 Illumina adaptor (for forward reads in paired-end sequencing) and a sample-specific 4-base EMOTE barcode (blue line and “XXX”, respectively) to index the molecules (different barcodes for the -RppH and + RppH pools). The barcode of the -RppH pool will designate molecules where the XRN1 treatments has been incomplete, and this information is incorporated into the in silico analysis (see below). g The barcoded DNA from various samples (and pools) can be mixed, and loaded directly into an Illumina HiSeq machine. Millions of 50 nt sequences are obtained, each of which will span the EMOTE barcode, both known and random sections of the Rp6 oligo (see Methods ), and it will reveal the first 20 nt of the native 5’-end of the ligated RNA molecule. These 20 nt are sufficient to map the vast majority of 5’-ends to a unique position on the small genomes of the bacteria in this study. However, longer Illumina reads (and thus longer mapping sequences) can be used if the TSSs are in repeated regions or if large-genome organisms, such as humans, are being examined. h The in silico pipeline input consists of stranded RNA-seq reads for one or multiple biological replicates in FASTQ format. Each replicate includes a FASTQ for the -RppH pool and another for the + RppH pool. i The FASTQ files go through EMOTE-conv software [ 51 ] that parses the reads, aligns them to the genome, and perform the quantification. Thus, for each genomic position we obtain the number of reads whose first nucleotide align at this genomic position, and on which strand it maps. The counts are further corrected for PCR biases by looking at the unique molecular identifiers (UMIs) sequences available in the unaligned part of the EMOTE read. j Quantification counts obtained for + RppH and -RppH pools are compared through a beta-binomial model that tests whether the identified 5’ ends in the + RppH pool is significantly enriched over the identified 5’ ends in the -RppH pool at a given position. The process results in a p-value that reflects our confidence in the genomic position to be enriched in the + RppH pool of the biological replicate. k The p-values of all the biological replicates are combined into a single p-value with Fisher’s method. l and m To correct the p-values for multiple testing across all genomic positions, the false discovery rate (FDR) is evaluated and only those with a FDR ≤ 0.01 are considered to be TSSs. Note also that for the FDR is only calculated for genomic positions with at least 5 detected ligation-events in at least one of the + RppH pools (UMI ≥ 5). n The TSSs then enter an annotation process that retrieve their surrounding sequence and downstream ORFs. TSSs separated by less than 5 bp are clustered together. Finally, to draw a global picture of operon structures, an independent detection of transcription terminators is operated with the software TransTermHP [ 39 ]. o Sequence of the RNA oligo Rp6 and a typical Illumina sequencing read from a TSS-EMOTE experiment. The Recognition Sequence serves as priming site for the PCR in panel F. UMI: The randomly incorporated nucleotides in the Rp6 oligo that serves to whether Illumina reads with identical Mapping Sequences originate from separate ligation events. CS: Control Sequence. EB: EMOTE barcode to index the Illumina reads. An asterisk indicates the 5’ nucleotide of the original RNA molecule

Techniques Used: In Silico, Purification, Ligation, Sequencing, Polymerase Chain Reaction, RNA Sequencing Assay, Software

28) Product Images from "A High-Throughput Method for Illumina RNA-Seq Library Preparation"

Article Title: A High-Throughput Method for Illumina RNA-Seq Library Preparation

Journal: Frontiers in Plant Science

doi: 10.3389/fpls.2012.00202

Outline of the high-throughput RNA-seq (HTR) library preparation . In short, frozen tissue samples are ground in the lysis buffer and mRNA is isolated from this using oligo dT beads (1). The mRNA is used to make first and second strands of cDNA (2) and this double stranded cDNA molecules are subsequently enzymatically fragmented (3). The ends of these molecules are repaired and an A nucleotide is added (4) to facilitate TA ligation of the barcoded adapters (5). The ligated samples are then enriched by amplification using adapter specific primers (6) and purified for sequencing.
Figure Legend Snippet: Outline of the high-throughput RNA-seq (HTR) library preparation . In short, frozen tissue samples are ground in the lysis buffer and mRNA is isolated from this using oligo dT beads (1). The mRNA is used to make first and second strands of cDNA (2) and this double stranded cDNA molecules are subsequently enzymatically fragmented (3). The ends of these molecules are repaired and an A nucleotide is added (4) to facilitate TA ligation of the barcoded adapters (5). The ligated samples are then enriched by amplification using adapter specific primers (6) and purified for sequencing.

Techniques Used: High Throughput Screening Assay, RNA Sequencing Assay, Lysis, Isolation, Ligation, Amplification, Purification, Sequencing

29) Product Images from "Quantitative Assessment of RNA-Protein Interactions with High Throughput Sequencing - RNA Affinity Profiling (HiTS-RAP)"

Article Title: Quantitative Assessment of RNA-Protein Interactions with High Throughput Sequencing - RNA Affinity Profiling (HiTS-RAP)

Journal: Nature protocols

doi: 10.1038/nprot.2015.074

DNA template and RNA transcript of HiTS-RAP. (A) Schematics of the DNA template used for HiTS-RAP and the resulting halted RNA transcript. DNA template encoding the RNA of interest (green) is flanked by the Illumina flowcell adaptor 1 (gray) and T7 RNA polymerase promoter (orange) upstream, and by the Illumina sequencing primer annealing site (purple), Tus-binding Ter site (red), and Illumina flowcell adaptor 2 downstream. Illumina flowcell adaptors 1 and 2 are required for cluster generation on Illumina GA flowcell. T7 RNA polymerase promoter is required for transcription of the RNA of interest. The Illumina sequencing primer is used for sequencing the DNA template of the RNA of interest and serves as a docking site for the T7 RNA polymerase when it is halted. Tus protein binds to Ter site and halts the transcribing RNA polymerase. Direction of transcription and sequencing are indicated by orange and purple arrows, respectively. Tus-bound Ter site that is non-permissive (halting) and permissive (read-through) to RNA polymerase are indicated by solid and open red triangles, respectively. The halted RNA transcript includes a triplet G derived from the T7 promoter, followed by the RNA of interest and some of the Illumina sequencing primer. The 3’-end of RNA transcript, indicated by a dashed line, is inaccessible. (B) Construction of DNA templates for HiTS-RAP. DNA template is constructed by PCR in two steps using 2 sets of nested oligos. Forward oligos introduce T7 promoter (step 1) and Illumina flowcell adaptor (step 2), whereas reverse oligos introduce Illumina sequencing primer (step 1), and Ter site and Illumina flowcell adaptor 2 (step 2). (C) Sequence of the HiTS-RAP DNA template for GFP aptamer. GFP aptamer encoding sequence is in green, and the rest of the sequences are colored as in (A). The transcription start site is indicated by +1 and a broken arrow. (D) Sequence of the halted RNA transcript from GFP aptamer template. Sequences are colored as in (C). Uppercase indicates the region of the halted RNA transcript that is accessible, and the lowercase indicates a region that is likely to be buried in T7 RNA polymerase and thus inaccessible by other proteins.
Figure Legend Snippet: DNA template and RNA transcript of HiTS-RAP. (A) Schematics of the DNA template used for HiTS-RAP and the resulting halted RNA transcript. DNA template encoding the RNA of interest (green) is flanked by the Illumina flowcell adaptor 1 (gray) and T7 RNA polymerase promoter (orange) upstream, and by the Illumina sequencing primer annealing site (purple), Tus-binding Ter site (red), and Illumina flowcell adaptor 2 downstream. Illumina flowcell adaptors 1 and 2 are required for cluster generation on Illumina GA flowcell. T7 RNA polymerase promoter is required for transcription of the RNA of interest. The Illumina sequencing primer is used for sequencing the DNA template of the RNA of interest and serves as a docking site for the T7 RNA polymerase when it is halted. Tus protein binds to Ter site and halts the transcribing RNA polymerase. Direction of transcription and sequencing are indicated by orange and purple arrows, respectively. Tus-bound Ter site that is non-permissive (halting) and permissive (read-through) to RNA polymerase are indicated by solid and open red triangles, respectively. The halted RNA transcript includes a triplet G derived from the T7 promoter, followed by the RNA of interest and some of the Illumina sequencing primer. The 3’-end of RNA transcript, indicated by a dashed line, is inaccessible. (B) Construction of DNA templates for HiTS-RAP. DNA template is constructed by PCR in two steps using 2 sets of nested oligos. Forward oligos introduce T7 promoter (step 1) and Illumina flowcell adaptor (step 2), whereas reverse oligos introduce Illumina sequencing primer (step 1), and Ter site and Illumina flowcell adaptor 2 (step 2). (C) Sequence of the HiTS-RAP DNA template for GFP aptamer. GFP aptamer encoding sequence is in green, and the rest of the sequences are colored as in (A). The transcription start site is indicated by +1 and a broken arrow. (D) Sequence of the halted RNA transcript from GFP aptamer template. Sequences are colored as in (C). Uppercase indicates the region of the halted RNA transcript that is accessible, and the lowercase indicates a region that is likely to be buried in T7 RNA polymerase and thus inaccessible by other proteins.

Techniques Used: Sequencing, Binding Assay, Derivative Assay, Construct, Polymerase Chain Reaction, Introduce

30) Product Images from "Loss of miR-29 in Myoblasts Contributes to Dystrophic Muscle Pathogenesis"

Article Title: Loss of miR-29 in Myoblasts Contributes to Dystrophic Muscle Pathogenesis

Journal: Molecular Therapy

doi: 10.1038/mt.2012.35

Overexpression of miR-29 in mdx muscles downregulates fibrotic genes . ( a ) Differentially expressed genes in mdx muscles injected with NC and miR-29 oligos as determined by mRNA-seq. X- and Y-axis represent the log2 based FPKM values for expressed genes in NC and miR-29 samples, respectively. ( b ) Over-represented GO terms by GO analysis of downregulated list of genes. BP, biological process; CC, cellular component; KEGG, Kyoto Encyclopedia of Genes and Genomes; SP_PIR, a database of protein super-family names. ( c ) Coverage plot showing a 56-kb region encompassing the ACTA2 (α-SMA) gene on chromosome (Chr) 19; the gene structure is shown in blue below the graph. ( d ) Expressions of collagen 1A1 (Col 1A1), collagen 1A2 (Col 1A2), collagen 3A1 (Col 3A1), α-smooth muscle actin (α-SMA) or vimentin (VIM) in NC or miR-29 injected muscles. ( e ) Expressions of the above genes in TA muscles injected with negative control (Anti-NC) or miR-29 inhibitor oligos (Anti-miR-29). FPKM, fragments per kilobase of transcript per million fragments mapped; GO, gene ontology; mRNA, messenger RNA; NC, negative control; TA, tibialis anterior.
Figure Legend Snippet: Overexpression of miR-29 in mdx muscles downregulates fibrotic genes . ( a ) Differentially expressed genes in mdx muscles injected with NC and miR-29 oligos as determined by mRNA-seq. X- and Y-axis represent the log2 based FPKM values for expressed genes in NC and miR-29 samples, respectively. ( b ) Over-represented GO terms by GO analysis of downregulated list of genes. BP, biological process; CC, cellular component; KEGG, Kyoto Encyclopedia of Genes and Genomes; SP_PIR, a database of protein super-family names. ( c ) Coverage plot showing a 56-kb region encompassing the ACTA2 (α-SMA) gene on chromosome (Chr) 19; the gene structure is shown in blue below the graph. ( d ) Expressions of collagen 1A1 (Col 1A1), collagen 1A2 (Col 1A2), collagen 3A1 (Col 3A1), α-smooth muscle actin (α-SMA) or vimentin (VIM) in NC or miR-29 injected muscles. ( e ) Expressions of the above genes in TA muscles injected with negative control (Anti-NC) or miR-29 inhibitor oligos (Anti-miR-29). FPKM, fragments per kilobase of transcript per million fragments mapped; GO, gene ontology; mRNA, messenger RNA; NC, negative control; TA, tibialis anterior.

Techniques Used: Over Expression, Injection, Negative Control

Loss of miR-29 promotes myoblasts transdifferentiation into myofibroblasts. ( a ) Expressions of Col 1A1, Col 1A2, and Col 3A1 in primary myoblasts freshly isolated from WT or mdx muscles. ( b ) Primary myoblasts from mdx muscles were transfected with NC or miR-29 oligos and examined for Col 1A1, Col 1A2, and Col 3A1 expressions. ( c ) WT or mutant Col 1A1, Col 1A2, or Col 3A1-3′UTR luciferase reporter constructs were transfected into mdx primary myoblasts with NC or miR-29 oligos. Luciferase activities were determined at 48 hours post-transfection. Relative luciferase unit (RLU) is shown with respect to NC cells where normalized luciferase values were set to 1. The data represents the average of three independent experiments ± SD. ( d ) Predicted binding between mmu-miR-29c and mouse 3′UTR of Mfap5. ( e ) WT or mutant Mfap5-3′UTR luciferase reporter constructs were transfected into C2C12 cells with NC or miR-29 oligos. Luciferase activities were determined as above. ( f ) Expressions of Mfap5 in primary myoblasts from WT or mdx muscles. ( g ) Primary myoblasts from mdx muscles were transfected with NC or miR-29 oligos and examined for Mfap5 expression. ( h ) Expressions of the Mfap5 mRNAs in mdx TA muscles injected with negative control (Anti-NC) or miR-29 inhibitor oligos (Anti-miR-29). ( i ) IF staining for MyoD (green) and α-SMA (red) were performed on cryosections of mdx TA muscles. DAPI (blue) staining was also performed to visualize the nuclei. A field with three types of cells is shown on the left: arrows, MyoD-/α-SMA+ cells; arrowhead, MyoD+/α-SMA- cells; asterisk, MyoD+/α-SMA+ cells. Higher magnification of one of the MyoD+/α-SMA+ cells is presented on the right. ( j ) TA muscles from mdx mice were injected with NC or miR-29 mimics oligos. Quantification of the MyoD+/SMA+ cells on the above muscles were performed on a minimal of 15 sections for each group. N = 5 mice per group. Data are plotted as mean ± SD.* P
Figure Legend Snippet: Loss of miR-29 promotes myoblasts transdifferentiation into myofibroblasts. ( a ) Expressions of Col 1A1, Col 1A2, and Col 3A1 in primary myoblasts freshly isolated from WT or mdx muscles. ( b ) Primary myoblasts from mdx muscles were transfected with NC or miR-29 oligos and examined for Col 1A1, Col 1A2, and Col 3A1 expressions. ( c ) WT or mutant Col 1A1, Col 1A2, or Col 3A1-3′UTR luciferase reporter constructs were transfected into mdx primary myoblasts with NC or miR-29 oligos. Luciferase activities were determined at 48 hours post-transfection. Relative luciferase unit (RLU) is shown with respect to NC cells where normalized luciferase values were set to 1. The data represents the average of three independent experiments ± SD. ( d ) Predicted binding between mmu-miR-29c and mouse 3′UTR of Mfap5. ( e ) WT or mutant Mfap5-3′UTR luciferase reporter constructs were transfected into C2C12 cells with NC or miR-29 oligos. Luciferase activities were determined as above. ( f ) Expressions of Mfap5 in primary myoblasts from WT or mdx muscles. ( g ) Primary myoblasts from mdx muscles were transfected with NC or miR-29 oligos and examined for Mfap5 expression. ( h ) Expressions of the Mfap5 mRNAs in mdx TA muscles injected with negative control (Anti-NC) or miR-29 inhibitor oligos (Anti-miR-29). ( i ) IF staining for MyoD (green) and α-SMA (red) were performed on cryosections of mdx TA muscles. DAPI (blue) staining was also performed to visualize the nuclei. A field with three types of cells is shown on the left: arrows, MyoD-/α-SMA+ cells; arrowhead, MyoD+/α-SMA- cells; asterisk, MyoD+/α-SMA+ cells. Higher magnification of one of the MyoD+/α-SMA+ cells is presented on the right. ( j ) TA muscles from mdx mice were injected with NC or miR-29 mimics oligos. Quantification of the MyoD+/SMA+ cells on the above muscles were performed on a minimal of 15 sections for each group. N = 5 mice per group. Data are plotted as mean ± SD.* P

Techniques Used: Isolation, Transfection, Mutagenesis, Luciferase, Construct, Binding Assay, Expressing, Injection, Negative Control, Staining, Mouse Assay

Systemic delivery of miR-29 oligos reduces fibrosis in mdx diaphragm . ( a ) Administration scheme for miR-29 injection. NC or miR-29 oligos formulated with liposome were injected into mdx mice through tail vein at day (d) 0, 4, and 7. Mice were killed and diaphragm muscles were harvested at day 21 for histology and immunostaining. n = 5 mice for each treatment group. ( b ) Expressions of miR-29 in various tissues collected at day 3 after the injection. ( c ) Trichrome staining of the fibrotic areas in NC and miR-29 injected diaphragm muscles. The positively stained areas were quantified with Image-Pro Plus from a minimum of 15 sections. ( d ) IHC staining of the above muscles with collagen 1. The positively stained areas were quantified as the above. ( e ) IF staining of the above muscle with collagen 1. ( f ) Expressions of Pax7, MyoD, myogenin, and YY1 mRNAs in the above treated diaphragm muscles. ( g ) H E staining of the above NC or miR-29 injected mdx muscles. ( h ) Damaged areas were identified as fibrotic and fatty areas and quantified as the above. ** P
Figure Legend Snippet: Systemic delivery of miR-29 oligos reduces fibrosis in mdx diaphragm . ( a ) Administration scheme for miR-29 injection. NC or miR-29 oligos formulated with liposome were injected into mdx mice through tail vein at day (d) 0, 4, and 7. Mice were killed and diaphragm muscles were harvested at day 21 for histology and immunostaining. n = 5 mice for each treatment group. ( b ) Expressions of miR-29 in various tissues collected at day 3 after the injection. ( c ) Trichrome staining of the fibrotic areas in NC and miR-29 injected diaphragm muscles. The positively stained areas were quantified with Image-Pro Plus from a minimum of 15 sections. ( d ) IHC staining of the above muscles with collagen 1. The positively stained areas were quantified as the above. ( e ) IF staining of the above muscle with collagen 1. ( f ) Expressions of Pax7, MyoD, myogenin, and YY1 mRNAs in the above treated diaphragm muscles. ( g ) H E staining of the above NC or miR-29 injected mdx muscles. ( h ) Damaged areas were identified as fibrotic and fatty areas and quantified as the above. ** P

Techniques Used: Injection, Mouse Assay, Immunostaining, Staining, Immunohistochemistry

miR-29 is downregulated in mdx myoblasts . ( a ) Expression of miR-29 in primary myoblasts from WT or mdx muscles. ( b ) Left: primary myoblasts from mdx muscles were kept growing (DM 0 hour) or differentiated (DM) for 7, 24, or 48 hours at which times cells were immunostained for MyHC. Right: positively stained cells were quantified. Numbers indicate the average number of MyHC positive cells counted from a minimum of 10 randomly chosen fields. Graphs are plotted as mean ± SD. Images were taken at 24 hours. ( c ) Expressions of MyHC or troponin RNAs in WT or mdx myoblasts differentiated for the indicated times. ( d ) Expression of miR-29 in mdx primary myoblasts transfected with NC or miR-29 oligos. ( e ) Left: the above transfected cells were differentiated for 48 hours at which time the cells were photographed under phase contrasts or immunostained for MyHC. Right: positively stained cells were quantified as in b . ( f ) Expressions of MyHC and troponin RNAs in NC or miR-29 transfected cells at different time points of differentiation. ( g ) mdx myoblasts were transfected with MyHC-Luc or TnI-Luc reporter plasmids and NC or miR-29 oligos. Cells were then differentiated for 48 hours at which time luciferase activities were determined. The data represent the average of three independent experiments ± SD. * P
Figure Legend Snippet: miR-29 is downregulated in mdx myoblasts . ( a ) Expression of miR-29 in primary myoblasts from WT or mdx muscles. ( b ) Left: primary myoblasts from mdx muscles were kept growing (DM 0 hour) or differentiated (DM) for 7, 24, or 48 hours at which times cells were immunostained for MyHC. Right: positively stained cells were quantified. Numbers indicate the average number of MyHC positive cells counted from a minimum of 10 randomly chosen fields. Graphs are plotted as mean ± SD. Images were taken at 24 hours. ( c ) Expressions of MyHC or troponin RNAs in WT or mdx myoblasts differentiated for the indicated times. ( d ) Expression of miR-29 in mdx primary myoblasts transfected with NC or miR-29 oligos. ( e ) Left: the above transfected cells were differentiated for 48 hours at which time the cells were photographed under phase contrasts or immunostained for MyHC. Right: positively stained cells were quantified as in b . ( f ) Expressions of MyHC and troponin RNAs in NC or miR-29 transfected cells at different time points of differentiation. ( g ) mdx myoblasts were transfected with MyHC-Luc or TnI-Luc reporter plasmids and NC or miR-29 oligos. Cells were then differentiated for 48 hours at which time luciferase activities were determined. The data represent the average of three independent experiments ± SD. * P

Techniques Used: Expressing, Staining, Transfection, Luciferase

31) Product Images from "sCLIP—an integrated platform to study RNA–protein interactomes in biomedical research: identification of CSTF2tau in alternative processing of small nuclear RNAs"

Article Title: sCLIP—an integrated platform to study RNA–protein interactomes in biomedical research: identification of CSTF2tau in alternative processing of small nuclear RNAs

Journal: Nucleic Acids Research

doi: 10.1093/nar/gkx152

sCLIP—a simplified platform for studying RNA–protein interactomes by using crosslinking immunoprecipitation (CLIP) sequencing with a highly sensitive and non-radioactive biochemistry for low input material. ( A ) Schematic overview of the sCLIP technique. Day 1: RNA–RBP interactions are preserved by in vivo UV-crosslinking. After crosslinking, the intact cells are lysed and the RNA not covered by crosslinked RBPs is partially digested. After immunoprecipitation (using antibodies against the RBP of interest; see necessary specificity controls in Supplementary Figure S1D and E ) an aliquot of the ribonucleoprotein (RNP) complexes is visualized by a non-radioactive labeling strategy (based on biotinylated ADP; see material and methods ; Day 2, Supplementary Figure S1F ). Following the IP the remaining material is digested with proteinase K and the bound RNA is released ( Supplementary Figure S1G ). Next, the RNA is polyadenylated and then reversely transcribed by using a modified oligo d(T) primer that harbors an in line and a random barcode along with a sequencing platform-compatible Illumina adaptor and a T7 promotor (Day 3). Following reverse transcription (RT) the cDNA is in vitro transcribed ( Supplementary Figure S1H ) and an Illumina adaptor is ligated to the mRNA 3΄end (Day 4). Finally, the amplified RNA is reversely transcribed and amplified with 10 cycles of PCR ( Supplementary Figure S1H ); afterward the libraries are subjected to high-throughput sequencing, and the sequencing data is analysed by an integrated sCLIP Data processing workflow (for further information see Supplementary Figures S1 and S2A and material and methods ; a detailed protocol of the procedure and the automated bioinformatics pipeline can be found in the supplementary informations ). ( B ) Reproducibility of binding sites between two replicates applying sCLIP against CSTF2tau (Pearson correlation between replicates was calculated to be 0.77; P -value = 2.2 × 10 −16 , for specificity controls see Supplementary Figure S1D–H ). ( C ) Out of all sequencing reads sCLIP delivers almost 60% of usable reads (green). In contrast the relative fraction of reads, which are either too short (cutoff
Figure Legend Snippet: sCLIP—a simplified platform for studying RNA–protein interactomes by using crosslinking immunoprecipitation (CLIP) sequencing with a highly sensitive and non-radioactive biochemistry for low input material. ( A ) Schematic overview of the sCLIP technique. Day 1: RNA–RBP interactions are preserved by in vivo UV-crosslinking. After crosslinking, the intact cells are lysed and the RNA not covered by crosslinked RBPs is partially digested. After immunoprecipitation (using antibodies against the RBP of interest; see necessary specificity controls in Supplementary Figure S1D and E ) an aliquot of the ribonucleoprotein (RNP) complexes is visualized by a non-radioactive labeling strategy (based on biotinylated ADP; see material and methods ; Day 2, Supplementary Figure S1F ). Following the IP the remaining material is digested with proteinase K and the bound RNA is released ( Supplementary Figure S1G ). Next, the RNA is polyadenylated and then reversely transcribed by using a modified oligo d(T) primer that harbors an in line and a random barcode along with a sequencing platform-compatible Illumina adaptor and a T7 promotor (Day 3). Following reverse transcription (RT) the cDNA is in vitro transcribed ( Supplementary Figure S1H ) and an Illumina adaptor is ligated to the mRNA 3΄end (Day 4). Finally, the amplified RNA is reversely transcribed and amplified with 10 cycles of PCR ( Supplementary Figure S1H ); afterward the libraries are subjected to high-throughput sequencing, and the sequencing data is analysed by an integrated sCLIP Data processing workflow (for further information see Supplementary Figures S1 and S2A and material and methods ; a detailed protocol of the procedure and the automated bioinformatics pipeline can be found in the supplementary informations ). ( B ) Reproducibility of binding sites between two replicates applying sCLIP against CSTF2tau (Pearson correlation between replicates was calculated to be 0.77; P -value = 2.2 × 10 −16 , for specificity controls see Supplementary Figure S1D–H ). ( C ) Out of all sequencing reads sCLIP delivers almost 60% of usable reads (green). In contrast the relative fraction of reads, which are either too short (cutoff

Techniques Used: Cross-linking Immunoprecipitation, Sequencing, In Vivo, Immunoprecipitation, Labeling, Modification, In Vitro, Amplification, Polymerase Chain Reaction, Next-Generation Sequencing, Binding Assay

32) Product Images from "tGBS® genotyping-by-sequencing enables reliable genotyping of heterozygous loci"

Article Title: tGBS® genotyping-by-sequencing enables reliable genotyping of heterozygous loci

Journal: Nucleic Acids Research

doi: 10.1093/nar/gkx853

Diagram of tGBS. Digestion . Genomic DNA is digested with two REs: NspI leaves a 3′overhang and BfuCI leaves a 5′ overhang. Ligation . Two distinct oligos are ligated to the complementary 3′ and 5′ overhangs. The oligo matching the 3′ overhang contains a sample-specific internal barcode sequence for sample identification. The oligo matching the 5′ overhang is universal and present in every reaction for later amplification. Selective PCR . Target sites are selected using a selective primer with variable selective bases (‘CA’) that match selected sequences in the digested genome fragments and a non-selective primer. When properly amplified, the selected sequence is complementary to the selective bases. Final PCR . Primers matching the amplification primer and the selective primer which contain the full Proton adaptor sequence are used for amplification of the final library. Final on-target sequence . The final sequence contains the 5′ Proton adaptor sequence, an internal barcode, the NspI RE site, the target molecule, selective bases, the BfuCI RE site and the 3′ Proton adaptor sequence. It is possible to adapt the tGBS protocol for sequencing on an Illumina instrument by redesigning the ligation oligos and PCR primers.
Figure Legend Snippet: Diagram of tGBS. Digestion . Genomic DNA is digested with two REs: NspI leaves a 3′overhang and BfuCI leaves a 5′ overhang. Ligation . Two distinct oligos are ligated to the complementary 3′ and 5′ overhangs. The oligo matching the 3′ overhang contains a sample-specific internal barcode sequence for sample identification. The oligo matching the 5′ overhang is universal and present in every reaction for later amplification. Selective PCR . Target sites are selected using a selective primer with variable selective bases (‘CA’) that match selected sequences in the digested genome fragments and a non-selective primer. When properly amplified, the selected sequence is complementary to the selective bases. Final PCR . Primers matching the amplification primer and the selective primer which contain the full Proton adaptor sequence are used for amplification of the final library. Final on-target sequence . The final sequence contains the 5′ Proton adaptor sequence, an internal barcode, the NspI RE site, the target molecule, selective bases, the BfuCI RE site and the 3′ Proton adaptor sequence. It is possible to adapt the tGBS protocol for sequencing on an Illumina instrument by redesigning the ligation oligos and PCR primers.

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

33) Product Images from "SOX2 suppresses the mobility of urothelial carcinoma by promoting the expression of S100A14"

Article Title: SOX2 suppresses the mobility of urothelial carcinoma by promoting the expression of S100A14

Journal: Biochemistry and Biophysics Reports

doi: 10.1016/j.bbrep.2016.06.016

SOX2 interacts with the 3′-UTR of the S100A14 mRNA in vivo . (A) The diagram depicts the annealing positions of the targeting oligomer for RNase H digestion and the primers for reverse transcription and PCR detection of the ORF and 3′-UTR fragments. (B) FLAG-SOX2 was transiently expressed in BFTC905 cells for 48 h, followed by the CLIP assay. The matrix-bound mRNA was incubated at 37 °C with RNase H in the absence of the targeting oligomer for 30 min. The mock-treated matrix-bound mRNA was then purified and reverse-transcribed. The full-length, ORF, and 3′ fragments of the S100A14 mRNA were detected by RT-PCR. (C) After immunoprecipitation, the matrix-bound mRNA was subject to oligomer-dependent RNase H digestion at 37 °C for 30 min. The ORF and 3′-UTR fragments of the S100A14 mRNA in the supernatant and bound to the matrix were recovered, reverse transcribed, and detected by semi-quantitative PCR.
Figure Legend Snippet: SOX2 interacts with the 3′-UTR of the S100A14 mRNA in vivo . (A) The diagram depicts the annealing positions of the targeting oligomer for RNase H digestion and the primers for reverse transcription and PCR detection of the ORF and 3′-UTR fragments. (B) FLAG-SOX2 was transiently expressed in BFTC905 cells for 48 h, followed by the CLIP assay. The matrix-bound mRNA was incubated at 37 °C with RNase H in the absence of the targeting oligomer for 30 min. The mock-treated matrix-bound mRNA was then purified and reverse-transcribed. The full-length, ORF, and 3′ fragments of the S100A14 mRNA were detected by RT-PCR. (C) After immunoprecipitation, the matrix-bound mRNA was subject to oligomer-dependent RNase H digestion at 37 °C for 30 min. The ORF and 3′-UTR fragments of the S100A14 mRNA in the supernatant and bound to the matrix were recovered, reverse transcribed, and detected by semi-quantitative PCR.

Techniques Used: In Vivo, Polymerase Chain Reaction, Cross-linking Immunoprecipitation, Incubation, Purification, Reverse Transcription Polymerase Chain Reaction, Immunoprecipitation, Real-time Polymerase Chain Reaction

34) Product Images from "Multiplexed Illumina sequencing libraries from picogram quantities of DNA"

Article Title: Multiplexed Illumina sequencing libraries from picogram quantities of DNA

Journal: BMC Genomics

doi: 10.1186/1471-2164-14-466

Oligonucleotide design and products of protocol. P5 and P7 are names given by Illumina to the oligo sequences that bind to the flow cell.
Figure Legend Snippet: Oligonucleotide design and products of protocol. P5 and P7 are names given by Illumina to the oligo sequences that bind to the flow cell.

Techniques Used: Flow Cytometry

35) Product Images from "Hierarchical regulation of the genome: global changes in nucleosome organization potentiate genome response"

Article Title: Hierarchical regulation of the genome: global changes in nucleosome organization potentiate genome response

Journal: Oncotarget

doi: 10.18632/oncotarget.6841

Mapping nucleosome distributions following KSHV reactivation using mTSS-seq technique and mTSS-seq validation A. Experimental design for mapping nucleosome distributions following KSHV reactivation using our newly developed mTSS-seq technique. B. Validation of mTSS-seq using quantitative PCR for both on-target regions of the genome (within the sequence-capture region, two-kb region centered on a TSS) and off-target regions of the genome (not within the sequence-capture region, outside the two-region centered on a TSS). Quantitative PCR was performed for on- and off-target regions of the genome for both RHOC and ITGA4. The y-axis shows C t values. On average, C t values between the on- and off-targets of the sequence-captured libraries differ by 10.5 cycles. C. The periodic occurrence of AA/TT/AT/TA dinucleotides was calculated for all nucleosomal-sized fragments (147-148 bp) at the 0 hour time point. The x-axis represents the distance from the dyad axis. The y-axis is the frequency of AA/TT/AT/TA dinucleotides. An A/T-containing dinucleotide periodicity is seen every 10 bp at the 0 hour time point. D. Alignment of the midpoint fragments (purple) from mTSS-seq to the human genome shown in the UCSC Genome Browser ( http://genome.ucsc.edu ). Zooming in 5000X on human chromosome 2 to a six-kb window with two-kb of midpoint fragments at 0 hour time point (purple lines), along with the sequence-capture oligos and previously-published human-nucleosome distribution map for cell line GM18508 (red lines, (Gaffney et al., 2012)) at the TSSs of PUS10 and PEX13. The x-axis is genomic location. The y-axis is scaled reads per million.
Figure Legend Snippet: Mapping nucleosome distributions following KSHV reactivation using mTSS-seq technique and mTSS-seq validation A. Experimental design for mapping nucleosome distributions following KSHV reactivation using our newly developed mTSS-seq technique. B. Validation of mTSS-seq using quantitative PCR for both on-target regions of the genome (within the sequence-capture region, two-kb region centered on a TSS) and off-target regions of the genome (not within the sequence-capture region, outside the two-region centered on a TSS). Quantitative PCR was performed for on- and off-target regions of the genome for both RHOC and ITGA4. The y-axis shows C t values. On average, C t values between the on- and off-targets of the sequence-captured libraries differ by 10.5 cycles. C. The periodic occurrence of AA/TT/AT/TA dinucleotides was calculated for all nucleosomal-sized fragments (147-148 bp) at the 0 hour time point. The x-axis represents the distance from the dyad axis. The y-axis is the frequency of AA/TT/AT/TA dinucleotides. An A/T-containing dinucleotide periodicity is seen every 10 bp at the 0 hour time point. D. Alignment of the midpoint fragments (purple) from mTSS-seq to the human genome shown in the UCSC Genome Browser ( http://genome.ucsc.edu ). Zooming in 5000X on human chromosome 2 to a six-kb window with two-kb of midpoint fragments at 0 hour time point (purple lines), along with the sequence-capture oligos and previously-published human-nucleosome distribution map for cell line GM18508 (red lines, (Gaffney et al., 2012)) at the TSSs of PUS10 and PEX13. The x-axis is genomic location. The y-axis is scaled reads per million.

Techniques Used: Real-time Polymerase Chain Reaction, Sequencing

36) Product Images from "A cost-effective RNA sequencing protocol for large-scale gene expression studies"

Article Title: A cost-effective RNA sequencing protocol for large-scale gene expression studies

Journal: Scientific Reports

doi: 10.1038/srep09570

Diagram of LM-Seq sample preparation protocol. Poly-A-tailed mRNA is isolated from total RNA using oligo-dT beads. Purified mRNA is then fragmented with heat in fragmentation buffer. First strand cDNA is then synthesized using random hexamer oligos containing partial Illumina 3′ adaptor sequence. After RNA removal, a modified oligo containing partial Illumina's 5′ adaptor is then ligated to the 5′ of the single stranded cDNA. The library is then amplified by PCR using oligos that contain full Illumina adaptor sequences and our in-house index sequences.
Figure Legend Snippet: Diagram of LM-Seq sample preparation protocol. Poly-A-tailed mRNA is isolated from total RNA using oligo-dT beads. Purified mRNA is then fragmented with heat in fragmentation buffer. First strand cDNA is then synthesized using random hexamer oligos containing partial Illumina 3′ adaptor sequence. After RNA removal, a modified oligo containing partial Illumina's 5′ adaptor is then ligated to the 5′ of the single stranded cDNA. The library is then amplified by PCR using oligos that contain full Illumina adaptor sequences and our in-house index sequences.

Techniques Used: Sample Prep, Isolation, Purification, Synthesized, Random Hexamer Labeling, Sequencing, Modification, Amplification, Polymerase Chain Reaction

37) Product Images from "High-throughput and quantitative genome-wide messenger RNA sequencing for molecular phenotyping"

Article Title: High-throughput and quantitative genome-wide messenger RNA sequencing for molecular phenotyping

Journal: BMC Genomics

doi: 10.1186/s12864-015-1788-6

DeTCT pipeline workflow. Between nine and 11 pairs of mutant and normal zebrafish embryos were collected from one clutch and RNA extracted. a Following DNaseI treatment and chemical fragmentation, molecules representing the 3′ end of transcripts were enriched by pulldown using an anchored biotinylated oligo dT primer attached to streptavidin magnetic beads (orange line). An RNA oligo matching part of the Illumina read 2 adapter (purple line) was ligated onto the 5′ end, the RNA eluted and annealed to an oligo comprising partial read 1 Illumina adapter (dark blue line) followed by 12 random bases (beige line), then an eight base indexing sequence specific to each sample (light blue line) and finally a 14 base anchored polyT sequence (grey line). After reverse transcription the Illumina adapter sequences were completed during library amplification. Libraries were quantified, pooled in equimolar amounts and sequenced by Illumina HiSeq 2500. b After decoding the indexing sequence, the trimmed zebrafish sequences (read 1 in green and read 2 in red) were mapped to the reference genome and duplicate reads were flagged. c The coordinate representing the transcript counting 3′ end (TC 3′ end) was predicted using the base immediately 3′ of the polyT sequence in read 1 (green dashed arrow and green curved line). After calling peaks using all mapped read 2s the resulting counts were associated with their respective sample (red curved line). The count data were used to identify differential transcript abundance between conditions using DESeq2 [ 28 ] and reported as a fold change with an adjusted p-value. The TC 3′ ends were matched to the closest Ensembl transcript 3′ ends on the same strand (black line). Gene list tables were produced and ordered by the lowest adjusted p-value. These gene lists were filtered for genes showing differential transcript abundance using the adjusted p-value and the proximity of the TC 3′ end and Ensembl gene end (typically adjusted p-value
Figure Legend Snippet: DeTCT pipeline workflow. Between nine and 11 pairs of mutant and normal zebrafish embryos were collected from one clutch and RNA extracted. a Following DNaseI treatment and chemical fragmentation, molecules representing the 3′ end of transcripts were enriched by pulldown using an anchored biotinylated oligo dT primer attached to streptavidin magnetic beads (orange line). An RNA oligo matching part of the Illumina read 2 adapter (purple line) was ligated onto the 5′ end, the RNA eluted and annealed to an oligo comprising partial read 1 Illumina adapter (dark blue line) followed by 12 random bases (beige line), then an eight base indexing sequence specific to each sample (light blue line) and finally a 14 base anchored polyT sequence (grey line). After reverse transcription the Illumina adapter sequences were completed during library amplification. Libraries were quantified, pooled in equimolar amounts and sequenced by Illumina HiSeq 2500. b After decoding the indexing sequence, the trimmed zebrafish sequences (read 1 in green and read 2 in red) were mapped to the reference genome and duplicate reads were flagged. c The coordinate representing the transcript counting 3′ end (TC 3′ end) was predicted using the base immediately 3′ of the polyT sequence in read 1 (green dashed arrow and green curved line). After calling peaks using all mapped read 2s the resulting counts were associated with their respective sample (red curved line). The count data were used to identify differential transcript abundance between conditions using DESeq2 [ 28 ] and reported as a fold change with an adjusted p-value. The TC 3′ ends were matched to the closest Ensembl transcript 3′ ends on the same strand (black line). Gene list tables were produced and ordered by the lowest adjusted p-value. These gene lists were filtered for genes showing differential transcript abundance using the adjusted p-value and the proximity of the TC 3′ end and Ensembl gene end (typically adjusted p-value

Techniques Used: Mutagenesis, Magnetic Beads, Sequencing, Amplification, Produced

38) Product Images from "Poly(A)-ClickSeq: click-chemistry for next-generation 3΄-end sequencing without RNA enrichment or fragmentation"

Article Title: Poly(A)-ClickSeq: click-chemistry for next-generation 3΄-end sequencing without RNA enrichment or fragmentation

Journal: Nucleic Acids Research

doi: 10.1093/nar/gkx286

Schematic overview of Poly(A)ClickSeq (PAC-seq). ( A ) RT-PCR is launched from a non-anchored Poly(T) primer containing a portion of the Illumina p7 adaptor. RT-PCR is performed in the presence of AzATP, AzGTP and AzCTP, but not AzTTP, thus only allowing chain termination to occur upstream of the poly(A) tail in the 3΄UTR. ( B ) 3΄-Azido-blocked cDNA fragments are ‘click-ligated’ to 5΄-hexynyl–functionalised DNA oligos containing the p5 Illumina adaptor. This yields triazole-linked ssDNA which can be PCR-amplified using primers to the p5 and p7 Illumina adaptors. ( C ) The cDNA library is analysed by gel electrophoresis and should consist of a smear of DNA products centered ∼200–300 bp. Appropriate cDNA fragment sizes are cut out of the gel and purified to yield a final library. ( D ) The final library consists of DNA fragments containing the Illumina p5 adaptor, a portion of the 3΄UTR, a stretch of As derived from both the RNA template and the poly(T) primer, and finally the p7 Illumina Indexing primer.
Figure Legend Snippet: Schematic overview of Poly(A)ClickSeq (PAC-seq). ( A ) RT-PCR is launched from a non-anchored Poly(T) primer containing a portion of the Illumina p7 adaptor. RT-PCR is performed in the presence of AzATP, AzGTP and AzCTP, but not AzTTP, thus only allowing chain termination to occur upstream of the poly(A) tail in the 3΄UTR. ( B ) 3΄-Azido-blocked cDNA fragments are ‘click-ligated’ to 5΄-hexynyl–functionalised DNA oligos containing the p5 Illumina adaptor. This yields triazole-linked ssDNA which can be PCR-amplified using primers to the p5 and p7 Illumina adaptors. ( C ) The cDNA library is analysed by gel electrophoresis and should consist of a smear of DNA products centered ∼200–300 bp. Appropriate cDNA fragment sizes are cut out of the gel and purified to yield a final library. ( D ) The final library consists of DNA fragments containing the Illumina p5 adaptor, a portion of the 3΄UTR, a stretch of As derived from both the RNA template and the poly(T) primer, and finally the p7 Illumina Indexing primer.

Techniques Used: Reverse Transcription Polymerase Chain Reaction, Polymerase Chain Reaction, Amplification, cDNA Library Assay, Nucleic Acid Electrophoresis, Purification, Derivative Assay

39) Product Images from "Highly selective retrieval of accurate DNA utilizing a pool of in situ-replicated DNA from multiple next-generation sequencing platforms"

Article Title: Highly selective retrieval of accurate DNA utilizing a pool of in situ-replicated DNA from multiple next-generation sequencing platforms

Journal: Nucleic Acids Research

doi: 10.1093/nar/gky016

Schematic of in situ replication of DNA molecules from next-generation sequencing (NGS) platforms and subsequent PCR-based retrieval of target sequences. ( A ) Process flow chart for PCR-based methods for the retrieval of error-free DNA targets from an NGS-replica pool. ( B ) Preparation strategy of 454 GS Junior sequencing-based retrieval. Combinatorial barcode-tagged (CBT) pools were processed from microarray-synthesized oligonucleotides and subsequently ligated to the sheared genomic DNA as flanking sequences. The library was replicated in a sealed NGS plate. ( C ) Preparation strategy of a pre-NGS pool (MiSeq and Ion Proton). The barcoded library (cgc50 pool) was directly synthesized on a microarray. ( D ) Schematic of library replication in a MiSeq flow cell. ( E ) Schematic of library replication using melt-off DNA in the Ion Proton system. This process could be automatically performed using an Ion OneTouch™ ES system.
Figure Legend Snippet: Schematic of in situ replication of DNA molecules from next-generation sequencing (NGS) platforms and subsequent PCR-based retrieval of target sequences. ( A ) Process flow chart for PCR-based methods for the retrieval of error-free DNA targets from an NGS-replica pool. ( B ) Preparation strategy of 454 GS Junior sequencing-based retrieval. Combinatorial barcode-tagged (CBT) pools were processed from microarray-synthesized oligonucleotides and subsequently ligated to the sheared genomic DNA as flanking sequences. The library was replicated in a sealed NGS plate. ( C ) Preparation strategy of a pre-NGS pool (MiSeq and Ion Proton). The barcoded library (cgc50 pool) was directly synthesized on a microarray. ( D ) Schematic of library replication in a MiSeq flow cell. ( E ) Schematic of library replication using melt-off DNA in the Ion Proton system. This process could be automatically performed using an Ion OneTouch™ ES system.

Techniques Used: In Situ, Next-Generation Sequencing, Polymerase Chain Reaction, Flow Cytometry, Sequencing, Microarray, Synthesized

40) Product Images from "tGBS® genotyping-by-sequencing enables reliable genotyping of heterozygous loci"

Article Title: tGBS® genotyping-by-sequencing enables reliable genotyping of heterozygous loci

Journal: Nucleic Acids Research

doi: 10.1093/nar/gkx853

Diagram of tGBS. Digestion . Genomic DNA is digested with two REs: NspI leaves a 3′overhang and BfuCI leaves a 5′ overhang. Ligation . Two distinct oligos are ligated to the complementary 3′ and 5′ overhangs. The oligo matching the 3′ overhang contains a sample-specific internal barcode sequence for sample identification. The oligo matching the 5′ overhang is universal and present in every reaction for later amplification. Selective PCR . Target sites are selected using a selective primer with variable selective bases (‘CA’) that match selected sequences in the digested genome fragments and a non-selective primer. When properly amplified, the selected sequence is complementary to the selective bases. Final PCR . Primers matching the amplification primer and the selective primer which contain the full Proton adaptor sequence are used for amplification of the final library. Final on-target sequence . The final sequence contains the 5′ Proton adaptor sequence, an internal barcode, the NspI RE site, the target molecule, selective bases, the BfuCI RE site and the 3′ Proton adaptor sequence. It is possible to adapt the tGBS protocol for sequencing on an Illumina instrument by redesigning the ligation oligos and PCR primers.
Figure Legend Snippet: Diagram of tGBS. Digestion . Genomic DNA is digested with two REs: NspI leaves a 3′overhang and BfuCI leaves a 5′ overhang. Ligation . Two distinct oligos are ligated to the complementary 3′ and 5′ overhangs. The oligo matching the 3′ overhang contains a sample-specific internal barcode sequence for sample identification. The oligo matching the 5′ overhang is universal and present in every reaction for later amplification. Selective PCR . Target sites are selected using a selective primer with variable selective bases (‘CA’) that match selected sequences in the digested genome fragments and a non-selective primer. When properly amplified, the selected sequence is complementary to the selective bases. Final PCR . Primers matching the amplification primer and the selective primer which contain the full Proton adaptor sequence are used for amplification of the final library. Final on-target sequence . The final sequence contains the 5′ Proton adaptor sequence, an internal barcode, the NspI RE site, the target molecule, selective bases, the BfuCI RE site and the 3′ Proton adaptor sequence. It is possible to adapt the tGBS protocol for sequencing on an Illumina instrument by redesigning the ligation oligos and PCR primers.

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

41) Product Images from "Investigation of Experimental Factors That Underlie BRCA1/2 mRNA Isoform Expression Variation: Recommendations for Utilizing Targeted RNA Sequencing to Evaluate Potential Spliceogenic Variants"

Article Title: Investigation of Experimental Factors That Underlie BRCA1/2 mRNA Isoform Expression Variation: Recommendations for Utilizing Targeted RNA Sequencing to Evaluate Potential Spliceogenic Variants

Journal: Frontiers in Oncology

doi: 10.3389/fonc.2018.00140

Relative expression of BRCA1 and BRCA2 mRNA isoforms in rare variant samples compared to controls. (A) Natural expression ranges of mRNA splice isoforms calculated from lymphoblastoid cell lines (LCLs) not containing any known spliceogenic variants in BRCA1 (A) and BRCA2 (C) . Colored symbols overlaid indicate the relative mRNA isoform expression in LCLs containing known BRCA1 (B) or BRCA2 (D) splice disrupting variants. Only mRNA splice isoforms that were detected by more than 10 reads in at least two controls were included. Mean and upper and lower limits shown for each isoform [SE (95%)].
Figure Legend Snippet: Relative expression of BRCA1 and BRCA2 mRNA isoforms in rare variant samples compared to controls. (A) Natural expression ranges of mRNA splice isoforms calculated from lymphoblastoid cell lines (LCLs) not containing any known spliceogenic variants in BRCA1 (A) and BRCA2 (C) . Colored symbols overlaid indicate the relative mRNA isoform expression in LCLs containing known BRCA1 (B) or BRCA2 (D) splice disrupting variants. Only mRNA splice isoforms that were detected by more than 10 reads in at least two controls were included. Mean and upper and lower limits shown for each isoform [SE (95%)].

Techniques Used: Expressing, Variant Assay

BRCA2 mRNA isoforms detected at six time points in an lymphoblastoid cell line (sample #7, Table S1 in Supplementary Material) treated with an nonsense-mediated decay inhibitor. A freeze–thaw process was undertaken after time points two and four. Three technical replicates are listed under each time point.
Figure Legend Snippet: BRCA2 mRNA isoforms detected at six time points in an lymphoblastoid cell line (sample #7, Table S1 in Supplementary Material) treated with an nonsense-mediated decay inhibitor. A freeze–thaw process was undertaken after time points two and four. Three technical replicates are listed under each time point.

Techniques Used:

42) Product Images from "RNAi-dependent Polycomb repression controls transposable elements in Tetrahymena"

Article Title: RNAi-dependent Polycomb repression controls transposable elements in Tetrahymena

Journal: Genes & Development

doi: 10.1101/gad.320796.118

The balance between ncRNA and mRNA production is a critical aspect of the balance between transcriptional silencing and activation. ( A ) IES-specific polyadenylated transcripts are only produced during developing MAC formation. (3) 3 h after mixing: meiosis, (6) 6 h after mixing: gametogenesis, (10) 10 h after mixing: developing MAC formation, (blue bar) IES. RNA-seq was performed after oligo-dT enrichment of polyadenylated transcripts. Note that essentially no RNA-seq reads were mapped to the IES region at the two early conjugation time points. (PM) Parental MAC, (DM) developing MAC, (OM) old MAC. ( B ) Percentage of RNA-seq reads mapped to consistently processed IESs, relative to total mappable reads. Note that IES-specific polyadenylated transcripts were abundantly produced during developing MAC formation (10 h after mixing) but rarely detected before that (3 and 6 h after mixing). ( C ) Localization of the transcriptional and cotranscriptional machineries in early (3 h after mixing) and late (10 h after mixing) conjugating cells. RPB3-HA, CBP20-HA, PRP19-HA, and THO2-HA cells were stained with an anti-HA antibody (red) and counterstained with DAPI (blue). Parental MAC (PM): green arrowhead, developing MAC (DM): green arrows, MIC: white arrowheads, old MAC (OM): white arrow. ( D ) Interaction between TWI1 and the transcriptional/cotranscriptional machineries. The designated cells were processed for crosslink-immunoprecipitation with the anti-HA antibody at late conjugation (10 h after mixing). The anti-HA and anti-TWI1 antibodies were used for immunoblotting. Note that similar amounts of bait proteins were recovered, as shown by the anti-HA immunoblotting. ( E ) Production of scnRNA from intronic as well as exonic regions. Distributions of polyadenylated transcripts (dotted lines) and scnRNA (solid lines) in introns and exons of IES-specific loci. Each intron is equally divided into 10 units from the 5′ to 3′ end; the two flanking exons are also equally divided into 10 units, respectively. The average RPM of each unit in all the loci were cumulated. Double counting for exons is avoided. ( F ) Alternative production of ncRNA and mRNA in the meiotic MIC and the developing MAC. In this simplified schematic, transcription from IES-specific loci is emphasized, while genic transcription from MDS is omitted. See text for details.
Figure Legend Snippet: The balance between ncRNA and mRNA production is a critical aspect of the balance between transcriptional silencing and activation. ( A ) IES-specific polyadenylated transcripts are only produced during developing MAC formation. (3) 3 h after mixing: meiosis, (6) 6 h after mixing: gametogenesis, (10) 10 h after mixing: developing MAC formation, (blue bar) IES. RNA-seq was performed after oligo-dT enrichment of polyadenylated transcripts. Note that essentially no RNA-seq reads were mapped to the IES region at the two early conjugation time points. (PM) Parental MAC, (DM) developing MAC, (OM) old MAC. ( B ) Percentage of RNA-seq reads mapped to consistently processed IESs, relative to total mappable reads. Note that IES-specific polyadenylated transcripts were abundantly produced during developing MAC formation (10 h after mixing) but rarely detected before that (3 and 6 h after mixing). ( C ) Localization of the transcriptional and cotranscriptional machineries in early (3 h after mixing) and late (10 h after mixing) conjugating cells. RPB3-HA, CBP20-HA, PRP19-HA, and THO2-HA cells were stained with an anti-HA antibody (red) and counterstained with DAPI (blue). Parental MAC (PM): green arrowhead, developing MAC (DM): green arrows, MIC: white arrowheads, old MAC (OM): white arrow. ( D ) Interaction between TWI1 and the transcriptional/cotranscriptional machineries. The designated cells were processed for crosslink-immunoprecipitation with the anti-HA antibody at late conjugation (10 h after mixing). The anti-HA and anti-TWI1 antibodies were used for immunoblotting. Note that similar amounts of bait proteins were recovered, as shown by the anti-HA immunoblotting. ( E ) Production of scnRNA from intronic as well as exonic regions. Distributions of polyadenylated transcripts (dotted lines) and scnRNA (solid lines) in introns and exons of IES-specific loci. Each intron is equally divided into 10 units from the 5′ to 3′ end; the two flanking exons are also equally divided into 10 units, respectively. The average RPM of each unit in all the loci were cumulated. Double counting for exons is avoided. ( F ) Alternative production of ncRNA and mRNA in the meiotic MIC and the developing MAC. In this simplified schematic, transcription from IES-specific loci is emphasized, while genic transcription from MDS is omitted. See text for details.

Techniques Used: Activation Assay, Produced, RNA Sequencing Assay, Conjugation Assay, Staining, Immunoprecipitation

43) Product Images from "The transcriptional activity of human Chromosome 22"

Article Title: The transcriptional activity of human Chromosome 22

Journal: Genes & Development

doi: 10.1101/gad.1055203

Differential hybridization mapping within positive PCR fragment sequences. ( A ) Hybridization to multiple 60-nt oligonucleotides positioned opposite an intron sequence annotated on the antisense strand. ( B ) Hybridization to oligonucleotides representing a predicted exon within an annotated intron on the sense strand. ( C ) Control spots showing differential hybridization to a known exon (1) located on the strand opposite an annotated intron and (2) whose expression was previously verified. NCBI/UCSC sequence coordinates are offset by 13 Mb to approximate the size of the unsequenced p arm.
Figure Legend Snippet: Differential hybridization mapping within positive PCR fragment sequences. ( A ) Hybridization to multiple 60-nt oligonucleotides positioned opposite an intron sequence annotated on the antisense strand. ( B ) Hybridization to oligonucleotides representing a predicted exon within an annotated intron on the sense strand. ( C ) Control spots showing differential hybridization to a known exon (1) located on the strand opposite an annotated intron and (2) whose expression was previously verified. NCBI/UCSC sequence coordinates are offset by 13 Mb to approximate the size of the unsequenced p arm.

Techniques Used: Hybridization, Polymerase Chain Reaction, Sequencing, Expressing

44) Product Images from "Ribonuclease L and metal-ion-independent endoribonuclease cleavage sites in host and viral RNAs"

Article Title: Ribonuclease L and metal-ion-independent endoribonuclease cleavage sites in host and viral RNAs

Journal: Nucleic Acids Research

doi: 10.1093/nar/gku118

Viral RNA fragments produced by RNase L and RNase A. HCV and PV RNAs were incubated with RNase L and RNase A to produce RNA fragments for 2′, 3′-cyclic phosphate cDNA synthesis and sequencing. Agarose gel electrophoresis and ethidium bromide staining revealed the size of viral RNA fragments. ( A ) Diagram of HCV and PV RNAs. HCV RNA is 9648 bases long. PV RNA is 7500 bases long. ( B ) Viral RNAs incubated with RNase L. HCV and PV RNAs were incubated with RNase L for 20 min in the absence of 2-5A (no 2-5A), or with RNase L and 2-5A for 0, 2.5, 5, 10 and 20 min. ( C ) Viral RNAs incubated with RNase A. HCV and PV RNAs were incubated for 20 min in the absence of RNase A (−), and the presence of RNase A for 0, 2.5, 5, 10 and 20 min.
Figure Legend Snippet: Viral RNA fragments produced by RNase L and RNase A. HCV and PV RNAs were incubated with RNase L and RNase A to produce RNA fragments for 2′, 3′-cyclic phosphate cDNA synthesis and sequencing. Agarose gel electrophoresis and ethidium bromide staining revealed the size of viral RNA fragments. ( A ) Diagram of HCV and PV RNAs. HCV RNA is 9648 bases long. PV RNA is 7500 bases long. ( B ) Viral RNAs incubated with RNase L. HCV and PV RNAs were incubated with RNase L for 20 min in the absence of 2-5A (no 2-5A), or with RNase L and 2-5A for 0, 2.5, 5, 10 and 20 min. ( C ) Viral RNAs incubated with RNase A. HCV and PV RNAs were incubated for 20 min in the absence of RNase A (−), and the presence of RNase A for 0, 2.5, 5, 10 and 20 min.

Techniques Used: Produced, Incubation, Sequencing, Agarose Gel Electrophoresis, Staining

Cleavage sites mapped onto rRNA secondary and tertiary structures. Secondary and tertiary structures from Anger et al. ( 42 ). ( A ) RNase L cleavage sites in 18S rRNA secondary structure. Portion of 18S rRNA structure highlighting the location of RNase L cleavage sites. ( B ) Location of RNase L cleavage sites in 80S ribosome tertiary structure. RNase L-dependent cleavage sites highlighted in red spheres. ( C ) 3′-end of 5.8S and 5S rRNAs. 3′-end of 5.8S and 5S rRNAs highlighted in orange spheres. ( D ) RNase L-independent cleavage sites. Some representative RNase L-independent cleavage sites highlighted in yellow spheres.
Figure Legend Snippet: Cleavage sites mapped onto rRNA secondary and tertiary structures. Secondary and tertiary structures from Anger et al. ( 42 ). ( A ) RNase L cleavage sites in 18S rRNA secondary structure. Portion of 18S rRNA structure highlighting the location of RNase L cleavage sites. ( B ) Location of RNase L cleavage sites in 80S ribosome tertiary structure. RNase L-dependent cleavage sites highlighted in red spheres. ( C ) 3′-end of 5.8S and 5S rRNAs. 3′-end of 5.8S and 5S rRNAs highlighted in orange spheres. ( D ) RNase L-independent cleavage sites. Some representative RNase L-independent cleavage sites highlighted in yellow spheres.

Techniques Used:

Endoribonuclease cleavage sites in rRNAs from W12 HeLa cells. RNAs from mock-infected and PV-infected W12 HeLa cells ( Figure 3 B) were used for 2′, 3′-cyclic phosphate cDNA synthesis and Illumina sequencing. The location and frequency of cleavage sites in 28S rRNA ( A ), 18S rRNA ( B ), 5.8S rRNA ( C ) and 5S rRNA ( D ) are shown for mock-infected and PV-infected RNA samples isolated at 8 hpa. X-axis: Nucleotide position of each RNA. Y-axis: Percentage of total UMIs at each cleavage site. Dinucleotides at the 3′-end of abundant RNA fragments are annotated at the corresponding positions in the graphs. The locations of GC-rich expansion segments are highlighted by light blue rectangles. RNase L cleavage sites are highlighted in red.
Figure Legend Snippet: Endoribonuclease cleavage sites in rRNAs from W12 HeLa cells. RNAs from mock-infected and PV-infected W12 HeLa cells ( Figure 3 B) were used for 2′, 3′-cyclic phosphate cDNA synthesis and Illumina sequencing. The location and frequency of cleavage sites in 28S rRNA ( A ), 18S rRNA ( B ), 5.8S rRNA ( C ) and 5S rRNA ( D ) are shown for mock-infected and PV-infected RNA samples isolated at 8 hpa. X-axis: Nucleotide position of each RNA. Y-axis: Percentage of total UMIs at each cleavage site. Dinucleotides at the 3′-end of abundant RNA fragments are annotated at the corresponding positions in the graphs. The locations of GC-rich expansion segments are highlighted by light blue rectangles. RNase L cleavage sites are highlighted in red.

Techniques Used: Infection, Sequencing, Isolation

45) Product Images from "Ribonuclease L and metal-ion-independent endoribonuclease cleavage sites in host and viral RNAs"

Article Title: Ribonuclease L and metal-ion-independent endoribonuclease cleavage sites in host and viral RNAs

Journal: Nucleic Acids Research

doi: 10.1093/nar/gku118

Viral RNA fragments produced by RNase L and RNase A. HCV and PV RNAs were incubated with RNase L and RNase A to produce RNA fragments for 2′, 3′-cyclic phosphate cDNA synthesis and sequencing. Agarose gel electrophoresis and ethidium bromide staining revealed the size of viral RNA fragments. ( A ) Diagram of HCV and PV RNAs. HCV RNA is 9648 bases long. PV RNA is 7500 bases long. ( B ) Viral RNAs incubated with RNase L. HCV and PV RNAs were incubated with RNase L for 20 min in the absence of 2-5A (no 2-5A), or with RNase L and 2-5A for 0, 2.5, 5, 10 and 20 min. ( C ) Viral RNAs incubated with RNase A. HCV and PV RNAs were incubated for 20 min in the absence of RNase A (−), and the presence of RNase A for 0, 2.5, 5, 10 and 20 min.
Figure Legend Snippet: Viral RNA fragments produced by RNase L and RNase A. HCV and PV RNAs were incubated with RNase L and RNase A to produce RNA fragments for 2′, 3′-cyclic phosphate cDNA synthesis and sequencing. Agarose gel electrophoresis and ethidium bromide staining revealed the size of viral RNA fragments. ( A ) Diagram of HCV and PV RNAs. HCV RNA is 9648 bases long. PV RNA is 7500 bases long. ( B ) Viral RNAs incubated with RNase L. HCV and PV RNAs were incubated with RNase L for 20 min in the absence of 2-5A (no 2-5A), or with RNase L and 2-5A for 0, 2.5, 5, 10 and 20 min. ( C ) Viral RNAs incubated with RNase A. HCV and PV RNAs were incubated for 20 min in the absence of RNase A (−), and the presence of RNase A for 0, 2.5, 5, 10 and 20 min.

Techniques Used: Produced, Incubation, Sequencing, Agarose Gel Electrophoresis, Staining

Cleavage sites mapped onto rRNA secondary and tertiary structures. Secondary and tertiary structures from Anger et al. ( 42 ). ( A ) RNase L cleavage sites in 18S rRNA secondary structure. Portion of 18S rRNA structure highlighting the location of RNase L cleavage sites. ( B ) Location of RNase L cleavage sites in 80S ribosome tertiary structure. RNase L-dependent cleavage sites highlighted in red spheres. ( C ) 3′-end of 5.8S and 5S rRNAs. 3′-end of 5.8S and 5S rRNAs highlighted in orange spheres. ( D ) RNase L-independent cleavage sites. Some representative RNase L-independent cleavage sites highlighted in yellow spheres.
Figure Legend Snippet: Cleavage sites mapped onto rRNA secondary and tertiary structures. Secondary and tertiary structures from Anger et al. ( 42 ). ( A ) RNase L cleavage sites in 18S rRNA secondary structure. Portion of 18S rRNA structure highlighting the location of RNase L cleavage sites. ( B ) Location of RNase L cleavage sites in 80S ribosome tertiary structure. RNase L-dependent cleavage sites highlighted in red spheres. ( C ) 3′-end of 5.8S and 5S rRNAs. 3′-end of 5.8S and 5S rRNAs highlighted in orange spheres. ( D ) RNase L-independent cleavage sites. Some representative RNase L-independent cleavage sites highlighted in yellow spheres.

Techniques Used:

Endoribonuclease cleavage sites in rRNAs from W12 HeLa cells. RNAs from mock-infected and PV-infected W12 HeLa cells ( Figure 3 B) were used for 2′, 3′-cyclic phosphate cDNA synthesis and Illumina sequencing. The location and frequency of cleavage sites in 28S rRNA ( A ), 18S rRNA ( B ), 5.8S rRNA ( C ) and 5S rRNA ( D ) are shown for mock-infected and PV-infected RNA samples isolated at 8 hpa. X-axis: Nucleotide position of each RNA. Y-axis: Percentage of total UMIs at each cleavage site. Dinucleotides at the 3′-end of abundant RNA fragments are annotated at the corresponding positions in the graphs. The locations of GC-rich expansion segments are highlighted by light blue rectangles. RNase L cleavage sites are highlighted in red.
Figure Legend Snippet: Endoribonuclease cleavage sites in rRNAs from W12 HeLa cells. RNAs from mock-infected and PV-infected W12 HeLa cells ( Figure 3 B) were used for 2′, 3′-cyclic phosphate cDNA synthesis and Illumina sequencing. The location and frequency of cleavage sites in 28S rRNA ( A ), 18S rRNA ( B ), 5.8S rRNA ( C ) and 5S rRNA ( D ) are shown for mock-infected and PV-infected RNA samples isolated at 8 hpa. X-axis: Nucleotide position of each RNA. Y-axis: Percentage of total UMIs at each cleavage site. Dinucleotides at the 3′-end of abundant RNA fragments are annotated at the corresponding positions in the graphs. The locations of GC-rich expansion segments are highlighted by light blue rectangles. RNase L cleavage sites are highlighted in red.

Techniques Used: Infection, Sequencing, Isolation

Related Articles

Amplification:

Article Title: The Draft Genome and Transcriptome of the Atlantic Horseshoe Crab, Limulus polyphemus
Article Snippet: RNA libraries were prepared using Illumina TruSeq RNA Sample Prep LT version 2 (RS-122-2001/2002). .. Illumina Reagents (Part #15012995) were used for PCR amplification.

Article Title: Nitrate application or P deficiency induce a decline in Medicago truncatula N2-fixation by similar changes in the nodule transcriptome
Article Snippet: Library preparation for RNAseq was performed using the TruSeq RNA Sample Preparation Kit (Illumina, Cat. N°RS-122-2002) starting from 500 ng of total RNA. .. The cDNA libraries were amplified and sequenced using the cBot and HiSeq2000 from Illumina (SR; 1 × 50 bp; 6 GB ca.

Article Title: H3K4 Methylation-Dependent Memory of Somatic Cell Identity Inhibits Reprogramming and Development of Nuclear Transfer Embryos
Article Snippet: .. Per sample, 500 ng RNA was used to generate a cDNA sequencing libraries using a Illumina TrueSeq kit (RS-122-2001), according to the manufacturer’s protocol using 12 PCR amplification cycles. .. cDNA synthesis and RTqPCR analysis cDNA synthesis was performed from the isolated RNAs using oligo dT(15) primers.

Article Title: Glass promotes the differentiation of neuronal and non-neuronal cell types in the Drosophila eye
Article Snippet: .. RNA-Seq library preps were made using the Illumina TruSeq RNA sample Prep Kit v2 (Cat #RS-122-2002), using 500 ng of total RNA as input, amplified by 12 cycles of PCR, and run on an Illumina 2500 (v4 chemistry), as single read 50. .. For each RNA-seq sample, sequence quality was assessed with FastQC ( http://www.bioinformatics.babraham.ac.uk/projects/fastqc ) and sequencing adapters were removed with Trimmomatic [ ].

Construct:

Article Title: Canonical and Noncanonical Actions of Arabidopsis Histone Deacetylases in Ribosomal RNA Processing
Article Snippet: .. RNA-seq and ChIP-seq libraries were constructed using a TruSeq RNA Library Preparation Kit (Illumina; RS-122-2002) and the Ovation Ultralow DR Multiplex System (NuGEN; 0330), respectively. ..

Article Title: Versatile approach for functional analysis of human proteins and efficient stable cell line generation using FLP-mediated recombination system
Article Snippet: RNA-seq libraries were constructed as previously described in Sultan et al., 2012 [ ] with minor modifications. .. Fragmentation and first strand cDNA synthesis were performed as in TruSeq RNA Library Prep kit v2 protocol (Illumina, RS-122-2001, instruction number 15026495 Rev.

Article Title: Cannabidiol induces antioxidant pathways in keratinocytes by targeting BACH1
Article Snippet: RNA was processed for high throughput sequencing using the Illumina TruSeq mRNA Sample Prep v2 kit (RS-122-2001). .. Transcriptome libraries were constructed by polyA purification.

Article Title: Transcriptome and Metabolite analysis reveal candidate genes of the cardiac glycoside biosynthetic pathway from Calotropis procera
Article Snippet: .. cDNA library construction for de novo Sequencing Tissues from 10 seedlings (3 month old whole seedling) were pooled together and RNA was isolated using Trizol reagent (Invitrogen Corp., Carlsbad, CA) and subsequently used to construct library with TruSeq RNA Sample preparation kit V2 (Illumina, Cat RS-122-2002) following manufactures protocol. .. 1 μg of total RNA by Qubit was enriched for Poly A using RNA Purification Beads.

Quantitation Assay:

Article Title: Nitrate application or P deficiency induce a decline in Medicago truncatula N2-fixation by similar changes in the nodule transcriptome
Article Snippet: Library preparation for RNAseq was performed using the TruSeq RNA Sample Preparation Kit (Illumina, Cat. N°RS-122-2002) starting from 500 ng of total RNA. .. Accurate quantitation of cDNA libraries was performed using the QuantiFluor™ dsDNA System (Promega, Mannheim, Germany).

Expressing:

Article Title: Canonical and Noncanonical Actions of Arabidopsis Histone Deacetylases in Ribosomal RNA Processing
Article Snippet: RNA-seq and ChIP-seq libraries were constructed using a TruSeq RNA Library Preparation Kit (Illumina; RS-122-2002) and the Ovation Ultralow DR Multiplex System (NuGEN; 0330), respectively. .. For RNA-seq, TopHat (2.0.8b) and Cufflinks (2.1.1) were used for differential expression analysis ( ).

Article Title: Genetic Inactivation of CD33 in Hematopoietic Stem Cells to Enable CAR T Cell Immunotherapy for Acute Myeloid Leukemia
Article Snippet: RNA-seq libraries were generated using TruSeq RNA library prep kit (Illumina, RS-122-2001/2) per the manufacturer’s instructions. .. Sequencing reads were aligned to the human genome (hg19) using RUM, and differential expression was analyzed using edgeR.

Modification:

Article Title: Versatile approach for functional analysis of human proteins and efficient stable cell line generation using FLP-mediated recombination system
Article Snippet: Fragmentation and first strand cDNA synthesis were performed as in TruSeq RNA Library Prep kit v2 protocol (Illumina, RS-122-2001, instruction number 15026495 Rev. .. Further steps: purification, end-repair, A-tailing and adapter ligation were performed as described in the previously mentioned TruSeq kit protocol with one modification: the first purification eluate was not decanted from the magnetic beads and subsequent steps were performed with the beads in solution.

Article Title: Transposable elements modulate human RNA abundance and splicing via specific RNA-protein interactions
Article Snippet: We extracted RNA using the buffer RWT/3X isopropanol modification detailed in ‘Appendix B: Optional On-Column DNAse Digestion…’ of the Qiagen miRNeasy® Mini Handbook. .. To remove ribosomal RNA, we fed ≥70 ng of input and fRIP RNA into the Ribo-Zero™ Magnetic Gold Kit (Epicentre; Madison, WI, USA; catalog number MRZG12324) followed by a cleanup using Agencourt RNAClean XP beads (Beckman Coulter; Brea, CA, USA; catalog number A63987) and elution with 19.5 μl of Elute, Prime, Fragment mix from the TruSeq RNA Sample Preparation Kit (Illumina; San Diego, CA, USA; catalog number RS-122-2001).

Ligation:

Article Title: The Draft Genome and Transcriptome of the Atlantic Horseshoe Crab, Limulus polyphemus
Article Snippet: RNA libraries were prepared using Illumina TruSeq RNA Sample Prep LT version 2 (RS-122-2001/2002). .. Complementary DNA libraries were created from the enriched RNA using Invitrogen Superscript II® per the manufacturers protocol. cDNA was fragmented, end repaired, and adenylated followed by a ligation of Illumina adaptor indices.

Article Title: Versatile approach for functional analysis of human proteins and efficient stable cell line generation using FLP-mediated recombination system
Article Snippet: Fragmentation and first strand cDNA synthesis were performed as in TruSeq RNA Library Prep kit v2 protocol (Illumina, RS-122-2001, instruction number 15026495 Rev. .. Further steps: purification, end-repair, A-tailing and adapter ligation were performed as described in the previously mentioned TruSeq kit protocol with one modification: the first purification eluate was not decanted from the magnetic beads and subsequent steps were performed with the beads in solution.

Generated:

Article Title: Genetic Inactivation of CD33 in Hematopoietic Stem Cells to Enable CAR T Cell Immunotherapy for Acute Myeloid Leukemia
Article Snippet: .. RNA-seq libraries were generated using TruSeq RNA library prep kit (Illumina, RS-122-2001/2) per the manufacturer’s instructions. .. The libraries were sequenced as 100bp single end using a HiSeq 2500 sequencer (Illumina).

Sequencing:

Article Title: Comparative RNA-seq Analysis in the Unsequenced Axolotl: The Oncogene Burst Highlights Early Gene Expression in the Blastema
Article Snippet: .. For ES and iPS cells, we prepared samples for sequencing using the Illumina TruSeq RNA Sample Preparation Kit v2 (RS-122-2001). .. The samples were quantitated with Life Technologies Qubit fluorometer (Q32857).

Article Title: Canonical and Noncanonical Actions of Arabidopsis Histone Deacetylases in Ribosomal RNA Processing
Article Snippet: Paragraph title: Library Construction, Sequencing, and Data Analysis ... RNA-seq and ChIP-seq libraries were constructed using a TruSeq RNA Library Preparation Kit (Illumina; RS-122-2002) and the Ovation Ultralow DR Multiplex System (NuGEN; 0330), respectively.

Article Title: The Polycomb-Dependent Epigenome Controls β Cell Dysfunction, Dedifferentiation, and Diabetes
Article Snippet: Total RNA extraction from purified islet is described above. mRNA from whole islets was used to generate libraries using Illumina TruSeq RNA Sample Prep v2 (RS-122-2001). .. All sequence data were performed in biological triplicates, each containing islets from 1-2 animals, at 2 x 50 bp length with high quality metrics ( > 20 Phred score) and nucleotide distribution.

Article Title: The Draft Genome and Transcriptome of the Atlantic Horseshoe Crab, Limulus polyphemus
Article Snippet: Paragraph title: 2.5. Transcriptome Illumina Paired-End Library Construction and Sequencing ... RNA libraries were prepared using Illumina TruSeq RNA Sample Prep LT version 2 (RS-122-2001/2002).

Article Title: Cannabidiol induces antioxidant pathways in keratinocytes by targeting BACH1
Article Snippet: RNA was processed for high throughput sequencing using the Illumina TruSeq mRNA Sample Prep v2 kit (RS-122-2001). .. In brief, 1 μg of total RNA from each sample was used to construct a cDNA library, followed by sequencing on the Illumina HiSeq 2500 system with single end 50 bp reads and ~30 millions of reads per sample.

Article Title: Genetic Inactivation of CD33 in Hematopoietic Stem Cells to Enable CAR T Cell Immunotherapy for Acute Myeloid Leukemia
Article Snippet: RNA-seq libraries were generated using TruSeq RNA library prep kit (Illumina, RS-122-2001/2) per the manufacturer’s instructions. .. Sequencing reads were aligned to the human genome (hg19) using RUM, and differential expression was analyzed using edgeR.

Article Title: Transcriptome and Metabolite analysis reveal candidate genes of the cardiac glycoside biosynthetic pathway from Calotropis procera
Article Snippet: .. cDNA library construction for de novo Sequencing Tissues from 10 seedlings (3 month old whole seedling) were pooled together and RNA was isolated using Trizol reagent (Invitrogen Corp., Carlsbad, CA) and subsequently used to construct library with TruSeq RNA Sample preparation kit V2 (Illumina, Cat RS-122-2002) following manufactures protocol. .. 1 μg of total RNA by Qubit was enriched for Poly A using RNA Purification Beads.

Article Title: Transposable elements modulate human RNA abundance and splicing via specific RNA-protein interactions
Article Snippet: To remove ribosomal RNA, we fed ≥70 ng of input and fRIP RNA into the Ribo-Zero™ Magnetic Gold Kit (Epicentre; Madison, WI, USA; catalog number MRZG12324) followed by a cleanup using Agencourt RNAClean XP beads (Beckman Coulter; Brea, CA, USA; catalog number A63987) and elution with 19.5 μl of Elute, Prime, Fragment mix from the TruSeq RNA Sample Preparation Kit (Illumina; San Diego, CA, USA; catalog number RS-122-2001). .. We pooled the resulting cDNA libraries and subjected them to paired-end sequencing on an Illumina HiSeq 2500 at a depth of 31 base pairs per read.

Article Title: H3K4 Methylation-Dependent Memory of Somatic Cell Identity Inhibits Reprogramming and Development of Nuclear Transfer Embryos
Article Snippet: .. Per sample, 500 ng RNA was used to generate a cDNA sequencing libraries using a Illumina TrueSeq kit (RS-122-2001), according to the manufacturer’s protocol using 12 PCR amplification cycles. .. cDNA synthesis and RTqPCR analysis cDNA synthesis was performed from the isolated RNAs using oligo dT(15) primers.

Article Title: Glass promotes the differentiation of neuronal and non-neuronal cell types in the Drosophila eye
Article Snippet: Library preparation and sequencing was carried out by the NYU Genome Technology Center. .. RNA-Seq library preps were made using the Illumina TruSeq RNA sample Prep Kit v2 (Cat #RS-122-2002), using 500 ng of total RNA as input, amplified by 12 cycles of PCR, and run on an Illumina 2500 (v4 chemistry), as single read 50.

Article Title: Identification of a unique gene expression signature in mercury and 2,3,7,8-tetrachlorodibenzo-p-dioxin co-exposed cells
Article Snippet: RNA-Seq libraries were prepared using an Illumina TruSeq RNA Sample Preparation Kit (RS-122-2002) according to the manufacturer’s protocol. .. Sequencing was performed as described earlier , using an Illumina HiSeq 2500 to obtain 50-nucleotide single-end reads.

ChIP-sequencing:

Article Title: Canonical and Noncanonical Actions of Arabidopsis Histone Deacetylases in Ribosomal RNA Processing
Article Snippet: .. RNA-seq and ChIP-seq libraries were constructed using a TruSeq RNA Library Preparation Kit (Illumina; RS-122-2002) and the Ovation Ultralow DR Multiplex System (NuGEN; 0330), respectively. ..

Article Title: The Polycomb-Dependent Epigenome Controls β Cell Dysfunction, Dedifferentiation, and Diabetes
Article Snippet: Paragraph title: Bulk RNA-seq and ChIP-seq ... Total RNA extraction from purified islet is described above. mRNA from whole islets was used to generate libraries using Illumina TruSeq RNA Sample Prep v2 (RS-122-2001).

Nucleic Acid Electrophoresis:

Article Title: Nitrate application or P deficiency induce a decline in Medicago truncatula N2-fixation by similar changes in the nodule transcriptome
Article Snippet: RNA was digested with RNAse-free DNAse (Qiagen, Hilden, Germany) and checked for integrity by capillary gel electrophoresis (Bioanalyzer, Agilent Technologies, Inc., Böblingen, Germany). .. Library preparation for RNAseq was performed using the TruSeq RNA Sample Preparation Kit (Illumina, Cat. N°RS-122-2002) starting from 500 ng of total RNA.

RNA Sequencing Assay:

Article Title: Recent polyploidization events in three Saccharum founding species
Article Snippet: Paragraph title: RNA‐Seq analysis ... A pair‐end library for LA Purple was made using Illumina® TruSeq™ RNA Sample Preparation Kit (RS‐122‐2001(2), Illumina).

Article Title: Canonical and Noncanonical Actions of Arabidopsis Histone Deacetylases in Ribosomal RNA Processing
Article Snippet: .. RNA-seq and ChIP-seq libraries were constructed using a TruSeq RNA Library Preparation Kit (Illumina; RS-122-2002) and the Ovation Ultralow DR Multiplex System (NuGEN; 0330), respectively. ..

Article Title: The Polycomb-Dependent Epigenome Controls β Cell Dysfunction, Dedifferentiation, and Diabetes
Article Snippet: Paragraph title: Bulk RNA-seq and ChIP-seq ... Total RNA extraction from purified islet is described above. mRNA from whole islets was used to generate libraries using Illumina TruSeq RNA Sample Prep v2 (RS-122-2001).

Article Title: Stable engineered vascular networks from human induced pluripotent stem cell-derived endothelial cells cultured in synthetic hydrogels
Article Snippet: Paragraph title: 2.9 RNA sequencing ... Total RNA was isolated using the RNeasy Kit (Qiagen) and included lysing in 350 µL RLT lysis buffer and the optional DNase treatment. cDNA libraries were prepared from 50 ng of total RNA and indexed with Illumina’s TruSeq RNA Sample Preparation Kit v2 (RS-122-2001 and RS-122-2002).

Article Title: Versatile approach for functional analysis of human proteins and efficient stable cell line generation using FLP-mediated recombination system
Article Snippet: RNA-seq libraries were constructed as previously described in Sultan et al., 2012 [ ] with minor modifications. .. Fragmentation and first strand cDNA synthesis were performed as in TruSeq RNA Library Prep kit v2 protocol (Illumina, RS-122-2001, instruction number 15026495 Rev.

Article Title: Cannabidiol induces antioxidant pathways in keratinocytes by targeting BACH1
Article Snippet: Paragraph title: RNA-Seq ... RNA was processed for high throughput sequencing using the Illumina TruSeq mRNA Sample Prep v2 kit (RS-122-2001).

Article Title: Genetic Inactivation of CD33 in Hematopoietic Stem Cells to Enable CAR T Cell Immunotherapy for Acute Myeloid Leukemia
Article Snippet: .. RNA-seq libraries were generated using TruSeq RNA library prep kit (Illumina, RS-122-2001/2) per the manufacturer’s instructions. .. The libraries were sequenced as 100bp single end using a HiSeq 2500 sequencer (Illumina).

Article Title: Glass promotes the differentiation of neuronal and non-neuronal cell types in the Drosophila eye
Article Snippet: .. RNA-Seq library preps were made using the Illumina TruSeq RNA sample Prep Kit v2 (Cat #RS-122-2002), using 500 ng of total RNA as input, amplified by 12 cycles of PCR, and run on an Illumina 2500 (v4 chemistry), as single read 50. .. For each RNA-seq sample, sequence quality was assessed with FastQC ( http://www.bioinformatics.babraham.ac.uk/projects/fastqc ) and sequencing adapters were removed with Trimmomatic [ ].

Article Title: Identification of a unique gene expression signature in mercury and 2,3,7,8-tetrachlorodibenzo-p-dioxin co-exposed cells
Article Snippet: .. RNA-Seq libraries were prepared using an Illumina TruSeq RNA Sample Preparation Kit (RS-122-2002) according to the manufacturer’s protocol. .. Sequencing was performed as described earlier , using an Illumina HiSeq 2500 to obtain 50-nucleotide single-end reads.

Magnetic Beads:

Article Title: The Draft Genome and Transcriptome of the Atlantic Horseshoe Crab, Limulus polyphemus
Article Snippet: RNA libraries were prepared using Illumina TruSeq RNA Sample Prep LT version 2 (RS-122-2001/2002). .. 1 μ g of total RNA was PolyA enriched using AMPure XP Magnetic Beads (Beckman Coulter #A63880).

Article Title: Versatile approach for functional analysis of human proteins and efficient stable cell line generation using FLP-mediated recombination system
Article Snippet: Fragmentation and first strand cDNA synthesis were performed as in TruSeq RNA Library Prep kit v2 protocol (Illumina, RS-122-2001, instruction number 15026495 Rev. .. Further steps: purification, end-repair, A-tailing and adapter ligation were performed as described in the previously mentioned TruSeq kit protocol with one modification: the first purification eluate was not decanted from the magnetic beads and subsequent steps were performed with the beads in solution.

Isolation:

Article Title: The Polycomb-Dependent Epigenome Controls β Cell Dysfunction, Dedifferentiation, and Diabetes
Article Snippet: Total RNA extraction from purified islet is described above. mRNA from whole islets was used to generate libraries using Illumina TruSeq RNA Sample Prep v2 (RS-122-2001). .. NEXSON ChIP-seq workflow ( ) is applied to freshly isolated mouse islets.

Article Title: Stable engineered vascular networks from human induced pluripotent stem cell-derived endothelial cells cultured in synthetic hydrogels
Article Snippet: .. Total RNA was isolated using the RNeasy Kit (Qiagen) and included lysing in 350 µL RLT lysis buffer and the optional DNase treatment. cDNA libraries were prepared from 50 ng of total RNA and indexed with Illumina’s TruSeq RNA Sample Preparation Kit v2 (RS-122-2001 and RS-122-2002). ..

Article Title: Versatile approach for functional analysis of human proteins and efficient stable cell line generation using FLP-mediated recombination system
Article Snippet: Paragraph title: RNA isolation, library construction and deep-sequencing ... Fragmentation and first strand cDNA synthesis were performed as in TruSeq RNA Library Prep kit v2 protocol (Illumina, RS-122-2001, instruction number 15026495 Rev.

Article Title: Cannabidiol induces antioxidant pathways in keratinocytes by targeting BACH1
Article Snippet: 2.3 RNA-Seq Total RNA was isolated from NHEK cells by Qiazol lysis reagent (Qiagen, Hilden, Germany) and purified with miRNeasy mini kit (Qiagen) following manufacturer's instructions. .. RNA was processed for high throughput sequencing using the Illumina TruSeq mRNA Sample Prep v2 kit (RS-122-2001).

Article Title: Genetic Inactivation of CD33 in Hematopoietic Stem Cells to Enable CAR T Cell Immunotherapy for Acute Myeloid Leukemia
Article Snippet: RNA was isolated from 5 matched samples of in vitro differentiated control/CD33 KO human HSPC using Ambion RiboPure RNA purification kit (Thermo Fisher Scientific, AM1924). .. RNA-seq libraries were generated using TruSeq RNA library prep kit (Illumina, RS-122-2001/2) per the manufacturer’s instructions.

Article Title: Transcriptome and Metabolite analysis reveal candidate genes of the cardiac glycoside biosynthetic pathway from Calotropis procera
Article Snippet: .. cDNA library construction for de novo Sequencing Tissues from 10 seedlings (3 month old whole seedling) were pooled together and RNA was isolated using Trizol reagent (Invitrogen Corp., Carlsbad, CA) and subsequently used to construct library with TruSeq RNA Sample preparation kit V2 (Illumina, Cat RS-122-2002) following manufactures protocol. .. 1 μg of total RNA by Qubit was enriched for Poly A using RNA Purification Beads.

Article Title: Glass promotes the differentiation of neuronal and non-neuronal cell types in the Drosophila eye
Article Snippet: RNA-Seq Larval tissue was isolated from 30 animals of each genotype in triplicate and RNA was extracted in Trizol (Invitrogen). .. RNA-Seq library preps were made using the Illumina TruSeq RNA sample Prep Kit v2 (Cat #RS-122-2002), using 500 ng of total RNA as input, amplified by 12 cycles of PCR, and run on an Illumina 2500 (v4 chemistry), as single read 50.

Article Title: Identification of a unique gene expression signature in mercury and 2,3,7,8-tetrachlorodibenzo-p-dioxin co-exposed cells
Article Snippet: Paragraph title: 2.2 RNA isolation and RNA-Seq ... RNA-Seq libraries were prepared using an Illumina TruSeq RNA Sample Preparation Kit (RS-122-2002) according to the manufacturer’s protocol.

Multiplex Assay:

Article Title: Comparative RNA-seq Analysis in the Unsequenced Axolotl: The Oncogene Burst Highlights Early Gene Expression in the Blastema
Article Snippet: For ES and iPS cells, we prepared samples for sequencing using the Illumina TruSeq RNA Sample Preparation Kit v2 (RS-122-2001). .. The HiSeq 2000 SR multiplex recipe was used.

Article Title: Canonical and Noncanonical Actions of Arabidopsis Histone Deacetylases in Ribosomal RNA Processing
Article Snippet: .. RNA-seq and ChIP-seq libraries were constructed using a TruSeq RNA Library Preparation Kit (Illumina; RS-122-2002) and the Ovation Ultralow DR Multiplex System (NuGEN; 0330), respectively. ..

Purification:

Article Title: The Polycomb-Dependent Epigenome Controls β Cell Dysfunction, Dedifferentiation, and Diabetes
Article Snippet: .. Total RNA extraction from purified islet is described above. mRNA from whole islets was used to generate libraries using Illumina TruSeq RNA Sample Prep v2 (RS-122-2001). .. The manufacturer’s recommendations were followed and the libraries were sequenced on an Illumina HiSeq 2500 sequencer.

Article Title: The Draft Genome and Transcriptome of the Atlantic Horseshoe Crab, Limulus polyphemus
Article Snippet: Transcriptome Illumina Paired-End Library Construction and Sequencing Extracted, purified RNA was quantified and analyzed using a Qubit 2.0 (Life Technology, Carlsbad CA) and an Agilent Bioanalyzer 2100 (Agilent Technology, Santa Clara, CA). .. RNA libraries were prepared using Illumina TruSeq RNA Sample Prep LT version 2 (RS-122-2001/2002).

Article Title: Nitrate application or P deficiency induce a decline in Medicago truncatula N2-fixation by similar changes in the nodule transcriptome
Article Snippet: RNA extraction, cDNA library preparation and RNAseq Total RNA was purified from 27 samples [(untreated controls, 4 h nitrate, P-deficiency) × (three biological replicates) × (nodules from three plants to be pooled for one biological treatment)] using the TRIzol protocol (Invitrogen, Frankfurt, Germany). .. Library preparation for RNAseq was performed using the TruSeq RNA Sample Preparation Kit (Illumina, Cat. N°RS-122-2002) starting from 500 ng of total RNA.

Article Title: Versatile approach for functional analysis of human proteins and efficient stable cell line generation using FLP-mediated recombination system
Article Snippet: Fragmentation and first strand cDNA synthesis were performed as in TruSeq RNA Library Prep kit v2 protocol (Illumina, RS-122-2001, instruction number 15026495 Rev. .. Further steps: purification, end-repair, A-tailing and adapter ligation were performed as described in the previously mentioned TruSeq kit protocol with one modification: the first purification eluate was not decanted from the magnetic beads and subsequent steps were performed with the beads in solution.

Article Title: Cannabidiol induces antioxidant pathways in keratinocytes by targeting BACH1
Article Snippet: 2.3 RNA-Seq Total RNA was isolated from NHEK cells by Qiazol lysis reagent (Qiagen, Hilden, Germany) and purified with miRNeasy mini kit (Qiagen) following manufacturer's instructions. .. RNA was processed for high throughput sequencing using the Illumina TruSeq mRNA Sample Prep v2 kit (RS-122-2001).

Article Title: Genetic Inactivation of CD33 in Hematopoietic Stem Cells to Enable CAR T Cell Immunotherapy for Acute Myeloid Leukemia
Article Snippet: RNA was isolated from 5 matched samples of in vitro differentiated control/CD33 KO human HSPC using Ambion RiboPure RNA purification kit (Thermo Fisher Scientific, AM1924). .. RNA-seq libraries were generated using TruSeq RNA library prep kit (Illumina, RS-122-2001/2) per the manufacturer’s instructions.

Article Title: Transcriptome and Metabolite analysis reveal candidate genes of the cardiac glycoside biosynthetic pathway from Calotropis procera
Article Snippet: cDNA library construction for de novo Sequencing Tissues from 10 seedlings (3 month old whole seedling) were pooled together and RNA was isolated using Trizol reagent (Invitrogen Corp., Carlsbad, CA) and subsequently used to construct library with TruSeq RNA Sample preparation kit V2 (Illumina, Cat RS-122-2002) following manufactures protocol. .. 1 μg of total RNA by Qubit was enriched for Poly A using RNA Purification Beads.

Article Title: Transposable elements modulate human RNA abundance and splicing via specific RNA-protein interactions
Article Snippet: Paragraph title: RNA purification and library construction ... To remove ribosomal RNA, we fed ≥70 ng of input and fRIP RNA into the Ribo-Zero™ Magnetic Gold Kit (Epicentre; Madison, WI, USA; catalog number MRZG12324) followed by a cleanup using Agencourt RNAClean XP beads (Beckman Coulter; Brea, CA, USA; catalog number A63987) and elution with 19.5 μl of Elute, Prime, Fragment mix from the TruSeq RNA Sample Preparation Kit (Illumina; San Diego, CA, USA; catalog number RS-122-2001).

Polymerase Chain Reaction:

Article Title: Comparative RNA-seq Analysis in the Unsequenced Axolotl: The Oncogene Burst Highlights Early Gene Expression in the Blastema
Article Snippet: Then 10 cycles of polymerase chain reaction (PCR) were performed to amplify the selected fragments using the Illumina supplied PCR primers and protocol. .. For ES and iPS cells, we prepared samples for sequencing using the Illumina TruSeq RNA Sample Preparation Kit v2 (RS-122-2001).

Article Title: Recent polyploidization events in three Saccharum founding species
Article Snippet: A pair‐end library for LA Purple was made using Illumina® TruSeq™ RNA Sample Preparation Kit (RS‐122‐2001(2), Illumina). .. A preprocessing quality control filter was imposed for both quality ( > 30) and depth of coverage ( > 7) to remove false positives due to PCR duplicates and low‐quality reads.

Article Title: The Draft Genome and Transcriptome of the Atlantic Horseshoe Crab, Limulus polyphemus
Article Snippet: RNA libraries were prepared using Illumina TruSeq RNA Sample Prep LT version 2 (RS-122-2001/2002). .. Illumina Reagents (Part #15012995) were used for PCR amplification.

Article Title: H3K4 Methylation-Dependent Memory of Somatic Cell Identity Inhibits Reprogramming and Development of Nuclear Transfer Embryos
Article Snippet: .. Per sample, 500 ng RNA was used to generate a cDNA sequencing libraries using a Illumina TrueSeq kit (RS-122-2001), according to the manufacturer’s protocol using 12 PCR amplification cycles. .. cDNA synthesis and RTqPCR analysis cDNA synthesis was performed from the isolated RNAs using oligo dT(15) primers.

Article Title: Glass promotes the differentiation of neuronal and non-neuronal cell types in the Drosophila eye
Article Snippet: .. RNA-Seq library preps were made using the Illumina TruSeq RNA sample Prep Kit v2 (Cat #RS-122-2002), using 500 ng of total RNA as input, amplified by 12 cycles of PCR, and run on an Illumina 2500 (v4 chemistry), as single read 50. .. For each RNA-seq sample, sequence quality was assessed with FastQC ( http://www.bioinformatics.babraham.ac.uk/projects/fastqc ) and sequencing adapters were removed with Trimmomatic [ ].

cDNA Library Assay:

Article Title: Nitrate application or P deficiency induce a decline in Medicago truncatula N2-fixation by similar changes in the nodule transcriptome
Article Snippet: Paragraph title: RNA extraction, cDNA library preparation and RNAseq ... Library preparation for RNAseq was performed using the TruSeq RNA Sample Preparation Kit (Illumina, Cat. N°RS-122-2002) starting from 500 ng of total RNA.

Article Title: Cannabidiol induces antioxidant pathways in keratinocytes by targeting BACH1
Article Snippet: RNA was processed for high throughput sequencing using the Illumina TruSeq mRNA Sample Prep v2 kit (RS-122-2001). .. In brief, 1 μg of total RNA from each sample was used to construct a cDNA library, followed by sequencing on the Illumina HiSeq 2500 system with single end 50 bp reads and ~30 millions of reads per sample.

Article Title: Transcriptome and Metabolite analysis reveal candidate genes of the cardiac glycoside biosynthetic pathway from Calotropis procera
Article Snippet: .. cDNA library construction for de novo Sequencing Tissues from 10 seedlings (3 month old whole seedling) were pooled together and RNA was isolated using Trizol reagent (Invitrogen Corp., Carlsbad, CA) and subsequently used to construct library with TruSeq RNA Sample preparation kit V2 (Illumina, Cat RS-122-2002) following manufactures protocol. .. 1 μg of total RNA by Qubit was enriched for Poly A using RNA Purification Beads.

Chromatin Immunoprecipitation:

Article Title: Nitrate application or P deficiency induce a decline in Medicago truncatula N2-fixation by similar changes in the nodule transcriptome
Article Snippet: Library preparation for RNAseq was performed using the TruSeq RNA Sample Preparation Kit (Illumina, Cat. N°RS-122-2002) starting from 500 ng of total RNA. .. The size range of the final cDNA libraries was determined by applying the DNA 1000 chip on the Bioanalyzer 2100 from Agilent (Böblingen, Germany) (280 bp).

RNA Extraction:

Article Title: The Polycomb-Dependent Epigenome Controls β Cell Dysfunction, Dedifferentiation, and Diabetes
Article Snippet: .. Total RNA extraction from purified islet is described above. mRNA from whole islets was used to generate libraries using Illumina TruSeq RNA Sample Prep v2 (RS-122-2001). .. The manufacturer’s recommendations were followed and the libraries were sequenced on an Illumina HiSeq 2500 sequencer.

Article Title: Nitrate application or P deficiency induce a decline in Medicago truncatula N2-fixation by similar changes in the nodule transcriptome
Article Snippet: Paragraph title: RNA extraction, cDNA library preparation and RNAseq ... Library preparation for RNAseq was performed using the TruSeq RNA Sample Preparation Kit (Illumina, Cat. N°RS-122-2002) starting from 500 ng of total RNA.

Sample Prep:

Article Title: Comparative RNA-seq Analysis in the Unsequenced Axolotl: The Oncogene Burst Highlights Early Gene Expression in the Blastema
Article Snippet: .. For ES and iPS cells, we prepared samples for sequencing using the Illumina TruSeq RNA Sample Preparation Kit v2 (RS-122-2001). .. The samples were quantitated with Life Technologies Qubit fluorometer (Q32857).

Article Title: Recent polyploidization events in three Saccharum founding species
Article Snippet: .. A pair‐end library for LA Purple was made using Illumina® TruSeq™ RNA Sample Preparation Kit (RS‐122‐2001(2), Illumina). .. The library samples were sequenced on the Illumina HiSeq 2000 with 120 cycles by the KECK centre in UIUC ( http://www.biotech.uiuc.edu/ ).

Article Title: The Polycomb-Dependent Epigenome Controls β Cell Dysfunction, Dedifferentiation, and Diabetes
Article Snippet: .. Total RNA extraction from purified islet is described above. mRNA from whole islets was used to generate libraries using Illumina TruSeq RNA Sample Prep v2 (RS-122-2001). .. The manufacturer’s recommendations were followed and the libraries were sequenced on an Illumina HiSeq 2500 sequencer.

Article Title: The Draft Genome and Transcriptome of the Atlantic Horseshoe Crab, Limulus polyphemus
Article Snippet: .. RNA libraries were prepared using Illumina TruSeq RNA Sample Prep LT version 2 (RS-122-2001/2002). .. 1 μ g of total RNA was PolyA enriched using AMPure XP Magnetic Beads (Beckman Coulter #A63880).

Article Title: Stable engineered vascular networks from human induced pluripotent stem cell-derived endothelial cells cultured in synthetic hydrogels
Article Snippet: .. Total RNA was isolated using the RNeasy Kit (Qiagen) and included lysing in 350 µL RLT lysis buffer and the optional DNase treatment. cDNA libraries were prepared from 50 ng of total RNA and indexed with Illumina’s TruSeq RNA Sample Preparation Kit v2 (RS-122-2001 and RS-122-2002). ..

Article Title: Nitrate application or P deficiency induce a decline in Medicago truncatula N2-fixation by similar changes in the nodule transcriptome
Article Snippet: .. Library preparation for RNAseq was performed using the TruSeq RNA Sample Preparation Kit (Illumina, Cat. N°RS-122-2002) starting from 500 ng of total RNA. .. Accurate quantitation of cDNA libraries was performed using the QuantiFluor™ dsDNA System (Promega, Mannheim, Germany).

Article Title: Cannabidiol induces antioxidant pathways in keratinocytes by targeting BACH1
Article Snippet: .. RNA was processed for high throughput sequencing using the Illumina TruSeq mRNA Sample Prep v2 kit (RS-122-2001). .. Transcriptome libraries were constructed by polyA purification.

Article Title: Transcriptome and Metabolite analysis reveal candidate genes of the cardiac glycoside biosynthetic pathway from Calotropis procera
Article Snippet: .. cDNA library construction for de novo Sequencing Tissues from 10 seedlings (3 month old whole seedling) were pooled together and RNA was isolated using Trizol reagent (Invitrogen Corp., Carlsbad, CA) and subsequently used to construct library with TruSeq RNA Sample preparation kit V2 (Illumina, Cat RS-122-2002) following manufactures protocol. .. 1 μg of total RNA by Qubit was enriched for Poly A using RNA Purification Beads.

Article Title: Transposable elements modulate human RNA abundance and splicing via specific RNA-protein interactions
Article Snippet: .. To remove ribosomal RNA, we fed ≥70 ng of input and fRIP RNA into the Ribo-Zero™ Magnetic Gold Kit (Epicentre; Madison, WI, USA; catalog number MRZG12324) followed by a cleanup using Agencourt RNAClean XP beads (Beckman Coulter; Brea, CA, USA; catalog number A63987) and elution with 19.5 μl of Elute, Prime, Fragment mix from the TruSeq RNA Sample Preparation Kit (Illumina; San Diego, CA, USA; catalog number RS-122-2001). ..

Article Title: Glass promotes the differentiation of neuronal and non-neuronal cell types in the Drosophila eye
Article Snippet: .. RNA-Seq library preps were made using the Illumina TruSeq RNA sample Prep Kit v2 (Cat #RS-122-2002), using 500 ng of total RNA as input, amplified by 12 cycles of PCR, and run on an Illumina 2500 (v4 chemistry), as single read 50. .. For each RNA-seq sample, sequence quality was assessed with FastQC ( http://www.bioinformatics.babraham.ac.uk/projects/fastqc ) and sequencing adapters were removed with Trimmomatic [ ].

Article Title: Identification of a unique gene expression signature in mercury and 2,3,7,8-tetrachlorodibenzo-p-dioxin co-exposed cells
Article Snippet: .. RNA-Seq libraries were prepared using an Illumina TruSeq RNA Sample Preparation Kit (RS-122-2002) according to the manufacturer’s protocol. .. Sequencing was performed as described earlier , using an Illumina HiSeq 2500 to obtain 50-nucleotide single-end reads.

In Vitro:

Article Title: Genetic Inactivation of CD33 in Hematopoietic Stem Cells to Enable CAR T Cell Immunotherapy for Acute Myeloid Leukemia
Article Snippet: RNA was isolated from 5 matched samples of in vitro differentiated control/CD33 KO human HSPC using Ambion RiboPure RNA purification kit (Thermo Fisher Scientific, AM1924). .. RNA-seq libraries were generated using TruSeq RNA library prep kit (Illumina, RS-122-2001/2) per the manufacturer’s instructions.

Next-Generation Sequencing:

Article Title: Cannabidiol induces antioxidant pathways in keratinocytes by targeting BACH1
Article Snippet: .. RNA was processed for high throughput sequencing using the Illumina TruSeq mRNA Sample Prep v2 kit (RS-122-2001). .. Transcriptome libraries were constructed by polyA purification.

Incubation:

Article Title: Versatile approach for functional analysis of human proteins and efficient stable cell line generation using FLP-mediated recombination system
Article Snippet: Fragmentation and first strand cDNA synthesis were performed as in TruSeq RNA Library Prep kit v2 protocol (Illumina, RS-122-2001, instruction number 15026495 Rev. .. For second strand synthesis, reaction mixtures were supplemented with 1 μl of 5x First Strand Synthesis Buffer (18080–085, Thermo Fisher Scientific), 15 μl 5x Second Strand Synthesis Buffer (10812–014, Thermo Fisher Scientific), 0.45 μl 50 mM MgCl, 1 μl 100 mM DTT, 2 μl of 10 mM dUNTP Mix (dATP, dGTP, dCTP, dUTP, 10 mM each, R0182, R0133, Thermo Fisher Scientific), water to 57 μl, 5 U E . coli DNA Ligase (M0205L, NEB), 20 U E . coli DNA Polymerase I (NEB, M0209L), 1 U RNase H (18021–071, Thermo Fisher Scientific), and incubated at 16°C for 2h.

Concentration Assay:

Article Title: Comparative RNA-seq Analysis in the Unsequenced Axolotl: The Oncogene Burst Highlights Early Gene Expression in the Blastema
Article Snippet: Samples were loaded on the flowcell cluster station at a concentration of 8 pM, and sequenced on the Illumina GAII. .. For ES and iPS cells, we prepared samples for sequencing using the Illumina TruSeq RNA Sample Preparation Kit v2 (RS-122-2001).

Lysis:

Article Title: Stable engineered vascular networks from human induced pluripotent stem cell-derived endothelial cells cultured in synthetic hydrogels
Article Snippet: .. Total RNA was isolated using the RNeasy Kit (Qiagen) and included lysing in 350 µL RLT lysis buffer and the optional DNase treatment. cDNA libraries were prepared from 50 ng of total RNA and indexed with Illumina’s TruSeq RNA Sample Preparation Kit v2 (RS-122-2001 and RS-122-2002). ..

Article Title: Cannabidiol induces antioxidant pathways in keratinocytes by targeting BACH1
Article Snippet: 2.3 RNA-Seq Total RNA was isolated from NHEK cells by Qiazol lysis reagent (Qiagen, Hilden, Germany) and purified with miRNeasy mini kit (Qiagen) following manufacturer's instructions. .. RNA was processed for high throughput sequencing using the Illumina TruSeq mRNA Sample Prep v2 kit (RS-122-2001).

Similar Products

  • Logo
  • About
  • News
  • Press Release
  • Team
  • Advisors
  • Partners
  • Contact
  • Bioz Stars
  • Bioz vStars
  • 91
    Illumina Inc oligo dt
    Comparison or mRNA-seq libraries from <t>oligo-dT</t> and Cap-captured mRNA. A-B. Reads from dT or Cap-capture prepared libraries were aligned to sequences consisting of the 5’ or 3’ 25% of Refseq <t>mRNAs.</t> Reads aligning to the 5’ and 3’ portions of the transcript are plotted. Blue line indicates a ratio of one. C. Reads from mRNA-seq libraries prepared using oligo-dT captured mRNAs from mitotic and interphase Xenopus egg extract were aligned to the Xenopus laevis Unigene database. Relative abundance of each mRNA in mitotic and interphase extracts is plotted. Red points highlight two-fold differences between mitotic and interphase samples. D. Same experiment as in panel C except that the mRNAs were purified using Cap-capture prior to library preparation. Red points highlight two-fold differences. E. Scatterplot of mRNA abundance in oligo-dT-captured and Cap-captured mRNA libraries. F. The ratio of reads per mRNA in mitotic and interphase extracts are plotted for oligo-dT captured mRNAs (X-axis, data from panel A) and cap-captured mRNAs (Y-axis data from panel B). The colored points correspond to mRNAs with known changes in poly-A tail length (cdk1, green; Eg2/aurora-a, orange; mos, red; Xlcl1, yellow). Quadrants highlight two-fold differences between samples.
    Oligo Dt, supplied by Illumina Inc, used in various techniques. Bioz Stars score: 91/100, based on 2 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/oligo dt/product/Illumina Inc
    Average 91 stars, based on 2 article reviews
    Price from $9.99 to $1999.99
    oligo dt - by Bioz Stars, 2020-02
    91/100 stars
      Buy from Supplier

    77
    Illumina Inc illumina oligo
    Quantification of synthetic double-stranded oligos. Four dilutions of two synthetic double-stranded oligos consisting of either the Ion Torrent “A” and “P1” adapter sequences ( a ) or the <t>Illumina</t> “i7” and “i5” adapter sequences ( b ) were quantified in duplicate with the NanoDrop ( ), Qubit ( ), Bioanalyzer ( ), GX Touch ( ), TapeStation ( ), and Fragment Analyzer ( ). The mean of the measured <t>oligo</t> concentrations were plotted against the concentrations given by the oligo supplier.
    Illumina Oligo, supplied by Illumina Inc, used in various techniques. Bioz Stars score: 77/100, based on 4 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/illumina oligo/product/Illumina Inc
    Average 77 stars, based on 4 article reviews
    Price from $9.99 to $1999.99
    illumina oligo - by Bioz Stars, 2020-02
    77/100 stars
      Buy from Supplier

    75
    Illumina Inc sequencing methods rnase l
    Viral RNA fragments produced by <t>RNase</t> L and RNase A. HCV and PV RNAs were incubated with RNase L and RNase A to produce RNA fragments for 2′, 3′-cyclic phosphate cDNA synthesis and sequencing. Agarose gel electrophoresis and ethidium bromide staining revealed the size of viral RNA fragments. ( A ) Diagram of HCV and PV RNAs. HCV RNA is 9648 bases long. PV RNA is 7500 bases long. ( B ) Viral RNAs incubated with RNase L. HCV and PV RNAs were incubated with RNase L for 20 min in the absence of 2-5A (no 2-5A), or with RNase L and 2-5A for 0, 2.5, 5, 10 and 20 min. ( C ) Viral RNAs incubated with RNase A. HCV and PV RNAs were incubated for 20 min in the absence of RNase A (−), and the presence of RNase A for 0, 2.5, 5, 10 and 20 min.
    Sequencing Methods Rnase L, supplied by Illumina Inc, used in various techniques. Bioz Stars score: 75/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/sequencing methods rnase l/product/Illumina Inc
    Average 75 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    sequencing methods rnase l - by Bioz Stars, 2020-02
    75/100 stars
      Buy from Supplier

    Image Search Results


    Comparison or mRNA-seq libraries from oligo-dT and Cap-captured mRNA. A-B. Reads from dT or Cap-capture prepared libraries were aligned to sequences consisting of the 5’ or 3’ 25% of Refseq mRNAs. Reads aligning to the 5’ and 3’ portions of the transcript are plotted. Blue line indicates a ratio of one. C. Reads from mRNA-seq libraries prepared using oligo-dT captured mRNAs from mitotic and interphase Xenopus egg extract were aligned to the Xenopus laevis Unigene database. Relative abundance of each mRNA in mitotic and interphase extracts is plotted. Red points highlight two-fold differences between mitotic and interphase samples. D. Same experiment as in panel C except that the mRNAs were purified using Cap-capture prior to library preparation. Red points highlight two-fold differences. E. Scatterplot of mRNA abundance in oligo-dT-captured and Cap-captured mRNA libraries. F. The ratio of reads per mRNA in mitotic and interphase extracts are plotted for oligo-dT captured mRNAs (X-axis, data from panel A) and cap-captured mRNAs (Y-axis data from panel B). The colored points correspond to mRNAs with known changes in poly-A tail length (cdk1, green; Eg2/aurora-a, orange; mos, red; Xlcl1, yellow). Quadrants highlight two-fold differences between samples.

    Journal: PLoS ONE

    Article Title: Combining Different mRNA Capture Methods to Analyze the Transcriptome: Analysis of the Xenopus laevis Transcriptome

    doi: 10.1371/journal.pone.0077700

    Figure Lengend Snippet: Comparison or mRNA-seq libraries from oligo-dT and Cap-captured mRNA. A-B. Reads from dT or Cap-capture prepared libraries were aligned to sequences consisting of the 5’ or 3’ 25% of Refseq mRNAs. Reads aligning to the 5’ and 3’ portions of the transcript are plotted. Blue line indicates a ratio of one. C. Reads from mRNA-seq libraries prepared using oligo-dT captured mRNAs from mitotic and interphase Xenopus egg extract were aligned to the Xenopus laevis Unigene database. Relative abundance of each mRNA in mitotic and interphase extracts is plotted. Red points highlight two-fold differences between mitotic and interphase samples. D. Same experiment as in panel C except that the mRNAs were purified using Cap-capture prior to library preparation. Red points highlight two-fold differences. E. Scatterplot of mRNA abundance in oligo-dT-captured and Cap-captured mRNA libraries. F. The ratio of reads per mRNA in mitotic and interphase extracts are plotted for oligo-dT captured mRNAs (X-axis, data from panel A) and cap-captured mRNAs (Y-axis data from panel B). The colored points correspond to mRNAs with known changes in poly-A tail length (cdk1, green; Eg2/aurora-a, orange; mos, red; Xlcl1, yellow). Quadrants highlight two-fold differences between samples.

    Article Snippet: To compare the mRNAs sampled by oligo-dT and Cap-capture methods we prepared Illumina libraries from Xenopus laevis egg extracts arrested in metaphase of meiosis II (labeled Mitosis or M for the remainder of the paper) and extracts induced to enter interphase (IF) by the addition of calcium, which mimics fertilization induced calcium release[ ].

    Techniques: Purification

    Poly-A tail analysis of selected mRNAs. mRNAs that exhibited changes in Mitosis:Interphase (M:IF) abundance ratios in oligo-dT-captured samples, but not in Cap-captured samples were analyzed for poly-A tail length using the ePAT assay and anchored TVN reverse transcription controls. A. Six mRNAs with high M:IF ratios (aurora-a, esco2, fbox5, stx11, march7, and hexim1) showed longer poly-A tails in mitotic extract. Two mRNA (setd8 and MGC83922) with a low M:IF ratio showed very modest changes in poly-A tail lengths between Mitosis and Interphase. Red asterix indicates the position of the prominent TVN PCR product that is quantified in B. B. The amount of PCR product contained in the TVN-RT PCR reactions (red asterix in A) were quantified. The ratio of the amount of PCR product in Mitotic to Interphase extracts is presented in the first line. The ratio of each mRNA in Mitotic and Interphase extracts as determined by RNA-seq is presented below the PCR derived ratios for comparison. In addition five mRNAs with high M:IF ratios (aurora-1, fbox5, stx11, march7, and hexim1) had increased amounts of minimal poly-A tail PCR products in mitosis compared to interphase in TVN controls (quantified below gel) while both setd8 and MGC88922 had higher levels of TVN PCR products in interphase compared to mitotic extracts TVN PCR products indicate mRNAs with poly-A tails of 18 As. Line traces of ePAT PCR reactions presented in panel A. Black lines indicate traces from mitotic extract and red lines indicate traces from interaphse extract. D. Semi-quantitative PCR for each of the mRNAs tested in A was performed on RNA from mitotic and interphase extracts. Random hexamers were used to prime reverse transcription for these reactions. A second experiment showing very similar results is present in Figure S2.

    Journal: PLoS ONE

    Article Title: Combining Different mRNA Capture Methods to Analyze the Transcriptome: Analysis of the Xenopus laevis Transcriptome

    doi: 10.1371/journal.pone.0077700

    Figure Lengend Snippet: Poly-A tail analysis of selected mRNAs. mRNAs that exhibited changes in Mitosis:Interphase (M:IF) abundance ratios in oligo-dT-captured samples, but not in Cap-captured samples were analyzed for poly-A tail length using the ePAT assay and anchored TVN reverse transcription controls. A. Six mRNAs with high M:IF ratios (aurora-a, esco2, fbox5, stx11, march7, and hexim1) showed longer poly-A tails in mitotic extract. Two mRNA (setd8 and MGC83922) with a low M:IF ratio showed very modest changes in poly-A tail lengths between Mitosis and Interphase. Red asterix indicates the position of the prominent TVN PCR product that is quantified in B. B. The amount of PCR product contained in the TVN-RT PCR reactions (red asterix in A) were quantified. The ratio of the amount of PCR product in Mitotic to Interphase extracts is presented in the first line. The ratio of each mRNA in Mitotic and Interphase extracts as determined by RNA-seq is presented below the PCR derived ratios for comparison. In addition five mRNAs with high M:IF ratios (aurora-1, fbox5, stx11, march7, and hexim1) had increased amounts of minimal poly-A tail PCR products in mitosis compared to interphase in TVN controls (quantified below gel) while both setd8 and MGC88922 had higher levels of TVN PCR products in interphase compared to mitotic extracts TVN PCR products indicate mRNAs with poly-A tails of 18 As. Line traces of ePAT PCR reactions presented in panel A. Black lines indicate traces from mitotic extract and red lines indicate traces from interaphse extract. D. Semi-quantitative PCR for each of the mRNAs tested in A was performed on RNA from mitotic and interphase extracts. Random hexamers were used to prime reverse transcription for these reactions. A second experiment showing very similar results is present in Figure S2.

    Article Snippet: To compare the mRNAs sampled by oligo-dT and Cap-capture methods we prepared Illumina libraries from Xenopus laevis egg extracts arrested in metaphase of meiosis II (labeled Mitosis or M for the remainder of the paper) and extracts induced to enter interphase (IF) by the addition of calcium, which mimics fertilization induced calcium release[ ].

    Techniques: Polymerase Chain Reaction, Reverse Transcription Polymerase Chain Reaction, RNA Sequencing Assay, Derivative Assay, Real-time Polymerase Chain Reaction

    Quantification of synthetic double-stranded oligos. Four dilutions of two synthetic double-stranded oligos consisting of either the Ion Torrent “A” and “P1” adapter sequences ( a ) or the Illumina “i7” and “i5” adapter sequences ( b ) were quantified in duplicate with the NanoDrop ( ), Qubit ( ), Bioanalyzer ( ), GX Touch ( ), TapeStation ( ), and Fragment Analyzer ( ). The mean of the measured oligo concentrations were plotted against the concentrations given by the oligo supplier.

    Journal: Scientific Reports

    Article Title: Quantification of massively parallel sequencing libraries – a comparative study of eight methods

    doi: 10.1038/s41598-018-19574-w

    Figure Lengend Snippet: Quantification of synthetic double-stranded oligos. Four dilutions of two synthetic double-stranded oligos consisting of either the Ion Torrent “A” and “P1” adapter sequences ( a ) or the Illumina “i7” and “i5” adapter sequences ( b ) were quantified in duplicate with the NanoDrop ( ), Qubit ( ), Bioanalyzer ( ), GX Touch ( ), TapeStation ( ), and Fragment Analyzer ( ). The mean of the measured oligo concentrations were plotted against the concentrations given by the oligo supplier.

    Article Snippet: The Illumina oligo was identical to the “i7” and “i5” adapter sequences.

    Techniques:

    Intragenic copy number detection and comparison to commercial panels. ( A ) Control genomic DNA samples were acquired from kConFab for sensitivity testing. Three of these samples included known exon duplications in BRCA1 , TP53 and MSH2 , which were assessed by the DeCON tool. Exons are numbered along the x-axis, and those of normal copy number are presented as blue dots. Amplifications are shown in red. The TP53 and AURKB genes are on opposing DNA strands hence the presence of the latter and its exons in this Figure. A similar genetic-overlap is observed for MSH2 to the left of the panel. ( B ) A commercially available pool of synthetic oligos against a normal genomic background was also obtained. Mutations were provided at variant allele frequencies (VAF) of 5–15% and 15–35%, or at germline frequencies. Presented are the number of detected and missed variants in our PV1 and PV2 panels relative to what was expected in AcroMetrix. This was compared to three other panels [AmpliSeq Cancer Hotspot Panel v2 (CHPv2), Illumina TruSeq Amplicon – Cancer Panel (TSCAP) and TruSight Tumor Panel 26 (TSTP)], the data for which were provided by the AcroMetrix manufacturer. Percent values on the right indicate the proportion of AcroMetrix variants actually targeted by the panels.

    Journal: Scientific Reports

    Article Title: Development and validation of a targeted gene sequencing panel for application to disparate cancers

    doi: 10.1038/s41598-019-52000-3

    Figure Lengend Snippet: Intragenic copy number detection and comparison to commercial panels. ( A ) Control genomic DNA samples were acquired from kConFab for sensitivity testing. Three of these samples included known exon duplications in BRCA1 , TP53 and MSH2 , which were assessed by the DeCON tool. Exons are numbered along the x-axis, and those of normal copy number are presented as blue dots. Amplifications are shown in red. The TP53 and AURKB genes are on opposing DNA strands hence the presence of the latter and its exons in this Figure. A similar genetic-overlap is observed for MSH2 to the left of the panel. ( B ) A commercially available pool of synthetic oligos against a normal genomic background was also obtained. Mutations were provided at variant allele frequencies (VAF) of 5–15% and 15–35%, or at germline frequencies. Presented are the number of detected and missed variants in our PV1 and PV2 panels relative to what was expected in AcroMetrix. This was compared to three other panels [AmpliSeq Cancer Hotspot Panel v2 (CHPv2), Illumina TruSeq Amplicon – Cancer Panel (TSCAP) and TruSight Tumor Panel 26 (TSTP)], the data for which were provided by the AcroMetrix manufacturer. Percent values on the right indicate the proportion of AcroMetrix variants actually targeted by the panels.

    Article Snippet: Oligos were captured with PV1 and sequenced on Illumina’s NextSeq500 platform in multiplexed pools.

    Techniques: Variant Assay, Amplification

    Viral RNA fragments produced by RNase L and RNase A. HCV and PV RNAs were incubated with RNase L and RNase A to produce RNA fragments for 2′, 3′-cyclic phosphate cDNA synthesis and sequencing. Agarose gel electrophoresis and ethidium bromide staining revealed the size of viral RNA fragments. ( A ) Diagram of HCV and PV RNAs. HCV RNA is 9648 bases long. PV RNA is 7500 bases long. ( B ) Viral RNAs incubated with RNase L. HCV and PV RNAs were incubated with RNase L for 20 min in the absence of 2-5A (no 2-5A), or with RNase L and 2-5A for 0, 2.5, 5, 10 and 20 min. ( C ) Viral RNAs incubated with RNase A. HCV and PV RNAs were incubated for 20 min in the absence of RNase A (−), and the presence of RNase A for 0, 2.5, 5, 10 and 20 min.

    Journal: Nucleic Acids Research

    Article Title: Ribonuclease L and metal-ion-independent endoribonuclease cleavage sites in host and viral RNAs

    doi: 10.1093/nar/gku118

    Figure Lengend Snippet: Viral RNA fragments produced by RNase L and RNase A. HCV and PV RNAs were incubated with RNase L and RNase A to produce RNA fragments for 2′, 3′-cyclic phosphate cDNA synthesis and sequencing. Agarose gel electrophoresis and ethidium bromide staining revealed the size of viral RNA fragments. ( A ) Diagram of HCV and PV RNAs. HCV RNA is 9648 bases long. PV RNA is 7500 bases long. ( B ) Viral RNAs incubated with RNase L. HCV and PV RNAs were incubated with RNase L for 20 min in the absence of 2-5A (no 2-5A), or with RNase L and 2-5A for 0, 2.5, 5, 10 and 20 min. ( C ) Viral RNAs incubated with RNase A. HCV and PV RNAs were incubated for 20 min in the absence of RNase A (−), and the presence of RNase A for 0, 2.5, 5, 10 and 20 min.

    Article Snippet: 2′, 3′-cyclic phosphate cDNA synthesis and Illumina sequencing methods RNase L, RNase A and other metal-ion–independent endoribonucleases target single-stranded regions of RNA, leaving 2′, 3′-cyclic phosphates at the end of RNA fragments ( ).

    Techniques: Produced, Incubation, Sequencing, Agarose Gel Electrophoresis, Staining

    Cleavage sites mapped onto rRNA secondary and tertiary structures. Secondary and tertiary structures from Anger et al. ( 42 ). ( A ) RNase L cleavage sites in 18S rRNA secondary structure. Portion of 18S rRNA structure highlighting the location of RNase L cleavage sites. ( B ) Location of RNase L cleavage sites in 80S ribosome tertiary structure. RNase L-dependent cleavage sites highlighted in red spheres. ( C ) 3′-end of 5.8S and 5S rRNAs. 3′-end of 5.8S and 5S rRNAs highlighted in orange spheres. ( D ) RNase L-independent cleavage sites. Some representative RNase L-independent cleavage sites highlighted in yellow spheres.

    Journal: Nucleic Acids Research

    Article Title: Ribonuclease L and metal-ion-independent endoribonuclease cleavage sites in host and viral RNAs

    doi: 10.1093/nar/gku118

    Figure Lengend Snippet: Cleavage sites mapped onto rRNA secondary and tertiary structures. Secondary and tertiary structures from Anger et al. ( 42 ). ( A ) RNase L cleavage sites in 18S rRNA secondary structure. Portion of 18S rRNA structure highlighting the location of RNase L cleavage sites. ( B ) Location of RNase L cleavage sites in 80S ribosome tertiary structure. RNase L-dependent cleavage sites highlighted in red spheres. ( C ) 3′-end of 5.8S and 5S rRNAs. 3′-end of 5.8S and 5S rRNAs highlighted in orange spheres. ( D ) RNase L-independent cleavage sites. Some representative RNase L-independent cleavage sites highlighted in yellow spheres.

    Article Snippet: 2′, 3′-cyclic phosphate cDNA synthesis and Illumina sequencing methods RNase L, RNase A and other metal-ion–independent endoribonucleases target single-stranded regions of RNA, leaving 2′, 3′-cyclic phosphates at the end of RNA fragments ( ).

    Techniques:

    Endoribonuclease cleavage sites in rRNAs from W12 HeLa cells. RNAs from mock-infected and PV-infected W12 HeLa cells ( Figure 3 B) were used for 2′, 3′-cyclic phosphate cDNA synthesis and Illumina sequencing. The location and frequency of cleavage sites in 28S rRNA ( A ), 18S rRNA ( B ), 5.8S rRNA ( C ) and 5S rRNA ( D ) are shown for mock-infected and PV-infected RNA samples isolated at 8 hpa. X-axis: Nucleotide position of each RNA. Y-axis: Percentage of total UMIs at each cleavage site. Dinucleotides at the 3′-end of abundant RNA fragments are annotated at the corresponding positions in the graphs. The locations of GC-rich expansion segments are highlighted by light blue rectangles. RNase L cleavage sites are highlighted in red.

    Journal: Nucleic Acids Research

    Article Title: Ribonuclease L and metal-ion-independent endoribonuclease cleavage sites in host and viral RNAs

    doi: 10.1093/nar/gku118

    Figure Lengend Snippet: Endoribonuclease cleavage sites in rRNAs from W12 HeLa cells. RNAs from mock-infected and PV-infected W12 HeLa cells ( Figure 3 B) were used for 2′, 3′-cyclic phosphate cDNA synthesis and Illumina sequencing. The location and frequency of cleavage sites in 28S rRNA ( A ), 18S rRNA ( B ), 5.8S rRNA ( C ) and 5S rRNA ( D ) are shown for mock-infected and PV-infected RNA samples isolated at 8 hpa. X-axis: Nucleotide position of each RNA. Y-axis: Percentage of total UMIs at each cleavage site. Dinucleotides at the 3′-end of abundant RNA fragments are annotated at the corresponding positions in the graphs. The locations of GC-rich expansion segments are highlighted by light blue rectangles. RNase L cleavage sites are highlighted in red.

    Article Snippet: 2′, 3′-cyclic phosphate cDNA synthesis and Illumina sequencing methods RNase L, RNase A and other metal-ion–independent endoribonucleases target single-stranded regions of RNA, leaving 2′, 3′-cyclic phosphates at the end of RNA fragments ( ).

    Techniques: Infection, Sequencing, Isolation