nebnext multiplex oligos Search Results


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  • 99
    New England Biolabs nebnext multiplex oligos
    Nebnext Multiplex Oligos, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 99/100, based on 3815 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Multiplex Oligos Oligonucleotides, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 99/100, based on 12481 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Nebnext Multiple Oligos, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 99/100, based on 15 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    New England Biolabs primer nebnext multiplex oligos
    Primer Nebnext Multiplex Oligos, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 99/100, based on 7 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    New England Biolabs dual index nebnext multiplex oligos
    Dual Index Nebnext Multiplex Oligos, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 99/100, based on 47 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    New England Biolabs nebnext multiplex oligos for illumina index primers set 2
    Nebnext Multiplex Oligos For Illumina Index Primers Set 2, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 99/100, based on 277 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Illumina Inc nebnext㠂 â multiplex oligos
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    New England Biolabs nebnext multiplex oligos for illumina methylated adaptor index primers set 1
    Nebnext Multiplex Oligos For Illumina Methylated Adaptor Index Primers Set 1, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 99/100, based on 65 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Beckman Coulter nebnext multiplex oligos for illumina index primers set 2
    Nebnext Multiplex Oligos For Illumina Index Primers Set 2, supplied by Beckman Coulter, used in various techniques. Bioz Stars score: 91/100, based on 2 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Illumina Inc nebnext multiplex oligos for illumina index primers set 1
    Nebnext Multiplex Oligos For Illumina Index Primers Set 1, supplied by Illumina Inc, used in various techniques. Bioz Stars score: 97/100, based on 48 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    New England Biolabs nebnext multioplex oligos
    Identification and global characterization of <t>DNA-associated</t> RNA. ( A ) Scatter plots showing the correlation between TriplexRNA isolated by DNA-IP and SPRI selection (left) and TriplexRNA (DNA-IP) and control RNA (middle, right). Pearson correlation coefficient ( R ) across 7148 genes overlapping peaks is shown. Green diagonal line x = y . Some representative genes that overlap TriplexRNAs and control RNAs are highlighted. ( B ) Pie charts depicting the genomic distribution of TriplexRNA (DNA-IP) compared to chromatin-associated and nuclear RNA peaks, excluding intronic and exonic gene regions. Upstream and downstream regions are defined within 2.5 kb proximity of the closest gene. ( C ) Pie chart showing classification of long noncoding RNAs that overlap TriplexRNA (DNA-IP), chromatin-associated and nuclear RNA. ( D ) Association of TriplexRNA (DNA-IP) and control RNA with ChromHMM promoter states and transcribed states. Active transcription start site (TssA), flanking active TSS (TssAFlnk), strong (Tx) and weak (TxWk) transcription regions are shown. ( E ) Left: Overlap of TriplexRNA (DNA-IP), chromatin-associated RNA and nuclear RNA with different classes of repeat elements. Right: Abundance of simple repeat subclasses. ( F ) Abundance of TriplexRNA (DNA-IP) and control RNAs overlapping super-enhancers in <t>HeLa</t> S3 cells. Data are from HeLa S3 cells. Adjusted P -values
    Nebnext Multioplex Oligos, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 99/100, based on 67 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Sage Science nebnext multiplex oligos for illumina index primers set 1
    Identification and global characterization of <t>DNA-associated</t> RNA. ( A ) Scatter plots showing the correlation between TriplexRNA isolated by DNA-IP and SPRI selection (left) and TriplexRNA (DNA-IP) and control RNA (middle, right). Pearson correlation coefficient ( R ) across 7148 genes overlapping peaks is shown. Green diagonal line x = y . Some representative genes that overlap TriplexRNAs and control RNAs are highlighted. ( B ) Pie charts depicting the genomic distribution of TriplexRNA (DNA-IP) compared to chromatin-associated and nuclear RNA peaks, excluding intronic and exonic gene regions. Upstream and downstream regions are defined within 2.5 kb proximity of the closest gene. ( C ) Pie chart showing classification of long noncoding RNAs that overlap TriplexRNA (DNA-IP), chromatin-associated and nuclear RNA. ( D ) Association of TriplexRNA (DNA-IP) and control RNA with ChromHMM promoter states and transcribed states. Active transcription start site (TssA), flanking active TSS (TssAFlnk), strong (Tx) and weak (TxWk) transcription regions are shown. ( E ) Left: Overlap of TriplexRNA (DNA-IP), chromatin-associated RNA and nuclear RNA with different classes of repeat elements. Right: Abundance of simple repeat subclasses. ( F ) Abundance of TriplexRNA (DNA-IP) and control RNAs overlapping super-enhancers in <t>HeLa</t> S3 cells. Data are from HeLa S3 cells. Adjusted P -values
    Nebnext Multiplex Oligos For Illumina Index Primers Set 1, supplied by Sage Science, used in various techniques. Bioz Stars score: 95/100, based on 9 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    New England Biolabs cas9 nuclease s pyogenes nebnext multiplex oligos
    Identification and global characterization of <t>DNA-associated</t> RNA. ( A ) Scatter plots showing the correlation between TriplexRNA isolated by DNA-IP and SPRI selection (left) and TriplexRNA (DNA-IP) and control RNA (middle, right). Pearson correlation coefficient ( R ) across 7148 genes overlapping peaks is shown. Green diagonal line x = y . Some representative genes that overlap TriplexRNAs and control RNAs are highlighted. ( B ) Pie charts depicting the genomic distribution of TriplexRNA (DNA-IP) compared to chromatin-associated and nuclear RNA peaks, excluding intronic and exonic gene regions. Upstream and downstream regions are defined within 2.5 kb proximity of the closest gene. ( C ) Pie chart showing classification of long noncoding RNAs that overlap TriplexRNA (DNA-IP), chromatin-associated and nuclear RNA. ( D ) Association of TriplexRNA (DNA-IP) and control RNA with ChromHMM promoter states and transcribed states. Active transcription start site (TssA), flanking active TSS (TssAFlnk), strong (Tx) and weak (TxWk) transcription regions are shown. ( E ) Left: Overlap of TriplexRNA (DNA-IP), chromatin-associated RNA and nuclear RNA with different classes of repeat elements. Right: Abundance of simple repeat subclasses. ( F ) Abundance of TriplexRNA (DNA-IP) and control RNAs overlapping super-enhancers in <t>HeLa</t> S3 cells. Data are from HeLa S3 cells. Adjusted P -values
    Cas9 Nuclease S Pyogenes Nebnext Multiplex Oligos, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 99/100, based on 4 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Illumina Inc nebnext multiplex oligos for illumina dual index primers set 1
    Identification and global characterization of <t>DNA-associated</t> RNA. ( A ) Scatter plots showing the correlation between TriplexRNA isolated by DNA-IP and SPRI selection (left) and TriplexRNA (DNA-IP) and control RNA (middle, right). Pearson correlation coefficient ( R ) across 7148 genes overlapping peaks is shown. Green diagonal line x = y . Some representative genes that overlap TriplexRNAs and control RNAs are highlighted. ( B ) Pie charts depicting the genomic distribution of TriplexRNA (DNA-IP) compared to chromatin-associated and nuclear RNA peaks, excluding intronic and exonic gene regions. Upstream and downstream regions are defined within 2.5 kb proximity of the closest gene. ( C ) Pie chart showing classification of long noncoding RNAs that overlap TriplexRNA (DNA-IP), chromatin-associated and nuclear RNA. ( D ) Association of TriplexRNA (DNA-IP) and control RNA with ChromHMM promoter states and transcribed states. Active transcription start site (TssA), flanking active TSS (TssAFlnk), strong (Tx) and weak (TxWk) transcription regions are shown. ( E ) Left: Overlap of TriplexRNA (DNA-IP), chromatin-associated RNA and nuclear RNA with different classes of repeat elements. Right: Abundance of simple repeat subclasses. ( F ) Abundance of TriplexRNA (DNA-IP) and control RNAs overlapping super-enhancers in <t>HeLa</t> S3 cells. Data are from HeLa S3 cells. Adjusted P -values
    Nebnext Multiplex Oligos For Illumina Dual Index Primers Set 1, supplied by Illumina Inc, used in various techniques. Bioz Stars score: 94/100, based on 4 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher nebnext multiplex oligos for illumina methylated adaptor index primers set 1
    Identification and global characterization of <t>DNA-associated</t> RNA. ( A ) Scatter plots showing the correlation between TriplexRNA isolated by DNA-IP and SPRI selection (left) and TriplexRNA (DNA-IP) and control RNA (middle, right). Pearson correlation coefficient ( R ) across 7148 genes overlapping peaks is shown. Green diagonal line x = y . Some representative genes that overlap TriplexRNAs and control RNAs are highlighted. ( B ) Pie charts depicting the genomic distribution of TriplexRNA (DNA-IP) compared to chromatin-associated and nuclear RNA peaks, excluding intronic and exonic gene regions. Upstream and downstream regions are defined within 2.5 kb proximity of the closest gene. ( C ) Pie chart showing classification of long noncoding RNAs that overlap TriplexRNA (DNA-IP), chromatin-associated and nuclear RNA. ( D ) Association of TriplexRNA (DNA-IP) and control RNA with ChromHMM promoter states and transcribed states. Active transcription start site (TssA), flanking active TSS (TssAFlnk), strong (Tx) and weak (TxWk) transcription regions are shown. ( E ) Left: Overlap of TriplexRNA (DNA-IP), chromatin-associated RNA and nuclear RNA with different classes of repeat elements. Right: Abundance of simple repeat subclasses. ( F ) Abundance of TriplexRNA (DNA-IP) and control RNAs overlapping super-enhancers in <t>HeLa</t> S3 cells. Data are from HeLa S3 cells. Adjusted P -values
    Nebnext Multiplex Oligos For Illumina Methylated Adaptor Index Primers Set 1, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 94/100, based on 8 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Illumina Inc multiplex oligos
    Identification and global characterization of <t>DNA-associated</t> RNA. ( A ) Scatter plots showing the correlation between TriplexRNA isolated by DNA-IP and SPRI selection (left) and TriplexRNA (DNA-IP) and control RNA (middle, right). Pearson correlation coefficient ( R ) across 7148 genes overlapping peaks is shown. Green diagonal line x = y . Some representative genes that overlap TriplexRNAs and control RNAs are highlighted. ( B ) Pie charts depicting the genomic distribution of TriplexRNA (DNA-IP) compared to chromatin-associated and nuclear RNA peaks, excluding intronic and exonic gene regions. Upstream and downstream regions are defined within 2.5 kb proximity of the closest gene. ( C ) Pie chart showing classification of long noncoding RNAs that overlap TriplexRNA (DNA-IP), chromatin-associated and nuclear RNA. ( D ) Association of TriplexRNA (DNA-IP) and control RNA with ChromHMM promoter states and transcribed states. Active transcription start site (TssA), flanking active TSS (TssAFlnk), strong (Tx) and weak (TxWk) transcription regions are shown. ( E ) Left: Overlap of TriplexRNA (DNA-IP), chromatin-associated RNA and nuclear RNA with different classes of repeat elements. Right: Abundance of simple repeat subclasses. ( F ) Abundance of TriplexRNA (DNA-IP) and control RNAs overlapping super-enhancers in <t>HeLa</t> S3 cells. Data are from HeLa S3 cells. Adjusted P -values
    Multiplex Oligos, supplied by Illumina Inc, used in various techniques. Bioz Stars score: 99/100, based on 329 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    New England Biolabs single cell low input rna library prep kit nebnext multiplex oligos
    Identification and global characterization of <t>DNA-associated</t> RNA. ( A ) Scatter plots showing the correlation between TriplexRNA isolated by DNA-IP and SPRI selection (left) and TriplexRNA (DNA-IP) and control RNA (middle, right). Pearson correlation coefficient ( R ) across 7148 genes overlapping peaks is shown. Green diagonal line x = y . Some representative genes that overlap TriplexRNAs and control RNAs are highlighted. ( B ) Pie charts depicting the genomic distribution of TriplexRNA (DNA-IP) compared to chromatin-associated and nuclear RNA peaks, excluding intronic and exonic gene regions. Upstream and downstream regions are defined within 2.5 kb proximity of the closest gene. ( C ) Pie chart showing classification of long noncoding RNAs that overlap TriplexRNA (DNA-IP), chromatin-associated and nuclear RNA. ( D ) Association of TriplexRNA (DNA-IP) and control RNA with ChromHMM promoter states and transcribed states. Active transcription start site (TssA), flanking active TSS (TssAFlnk), strong (Tx) and weak (TxWk) transcription regions are shown. ( E ) Left: Overlap of TriplexRNA (DNA-IP), chromatin-associated RNA and nuclear RNA with different classes of repeat elements. Right: Abundance of simple repeat subclasses. ( F ) Abundance of TriplexRNA (DNA-IP) and control RNAs overlapping super-enhancers in <t>HeLa</t> S3 cells. Data are from HeLa S3 cells. Adjusted P -values
    Single Cell Low Input Rna Library Prep Kit Nebnext Multiplex Oligos, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 99/100, based on 4 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    New England Biolabs nebnext multiplex small rna library prep set for illumina 1 12
    Identification and global characterization of <t>DNA-associated</t> RNA. ( A ) Scatter plots showing the correlation between TriplexRNA isolated by DNA-IP and SPRI selection (left) and TriplexRNA (DNA-IP) and control RNA (middle, right). Pearson correlation coefficient ( R ) across 7148 genes overlapping peaks is shown. Green diagonal line x = y . Some representative genes that overlap TriplexRNAs and control RNAs are highlighted. ( B ) Pie charts depicting the genomic distribution of TriplexRNA (DNA-IP) compared to chromatin-associated and nuclear RNA peaks, excluding intronic and exonic gene regions. Upstream and downstream regions are defined within 2.5 kb proximity of the closest gene. ( C ) Pie chart showing classification of long noncoding RNAs that overlap TriplexRNA (DNA-IP), chromatin-associated and nuclear RNA. ( D ) Association of TriplexRNA (DNA-IP) and control RNA with ChromHMM promoter states and transcribed states. Active transcription start site (TssA), flanking active TSS (TssAFlnk), strong (Tx) and weak (TxWk) transcription regions are shown. ( E ) Left: Overlap of TriplexRNA (DNA-IP), chromatin-associated RNA and nuclear RNA with different classes of repeat elements. Right: Abundance of simple repeat subclasses. ( F ) Abundance of TriplexRNA (DNA-IP) and control RNAs overlapping super-enhancers in <t>HeLa</t> S3 cells. Data are from HeLa S3 cells. Adjusted P -values
    Nebnext Multiplex Small Rna Library Prep Set For Illumina 1 12, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 99/100, based on 48 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Identification and global characterization of <t>DNA-associated</t> RNA. ( A ) Scatter plots showing the correlation between TriplexRNA isolated by DNA-IP and SPRI selection (left) and TriplexRNA (DNA-IP) and control RNA (middle, right). Pearson correlation coefficient ( R ) across 7148 genes overlapping peaks is shown. Green diagonal line x = y . Some representative genes that overlap TriplexRNAs and control RNAs are highlighted. ( B ) Pie charts depicting the genomic distribution of TriplexRNA (DNA-IP) compared to chromatin-associated and nuclear RNA peaks, excluding intronic and exonic gene regions. Upstream and downstream regions are defined within 2.5 kb proximity of the closest gene. ( C ) Pie chart showing classification of long noncoding RNAs that overlap TriplexRNA (DNA-IP), chromatin-associated and nuclear RNA. ( D ) Association of TriplexRNA (DNA-IP) and control RNA with ChromHMM promoter states and transcribed states. Active transcription start site (TssA), flanking active TSS (TssAFlnk), strong (Tx) and weak (TxWk) transcription regions are shown. ( E ) Left: Overlap of TriplexRNA (DNA-IP), chromatin-associated RNA and nuclear RNA with different classes of repeat elements. Right: Abundance of simple repeat subclasses. ( F ) Abundance of TriplexRNA (DNA-IP) and control RNAs overlapping super-enhancers in <t>HeLa</t> S3 cells. Data are from HeLa S3 cells. Adjusted P -values
    Nebnext Multiplex Small Rna Library Prep Set For Illumina Set 2, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 99/100, based on 5 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    New England Biolabs nebnext poly a mrna magnetic isolation module
    Identification and global characterization of <t>DNA-associated</t> RNA. ( A ) Scatter plots showing the correlation between TriplexRNA isolated by DNA-IP and SPRI selection (left) and TriplexRNA (DNA-IP) and control RNA (middle, right). Pearson correlation coefficient ( R ) across 7148 genes overlapping peaks is shown. Green diagonal line x = y . Some representative genes that overlap TriplexRNAs and control RNAs are highlighted. ( B ) Pie charts depicting the genomic distribution of TriplexRNA (DNA-IP) compared to chromatin-associated and nuclear RNA peaks, excluding intronic and exonic gene regions. Upstream and downstream regions are defined within 2.5 kb proximity of the closest gene. ( C ) Pie chart showing classification of long noncoding RNAs that overlap TriplexRNA (DNA-IP), chromatin-associated and nuclear RNA. ( D ) Association of TriplexRNA (DNA-IP) and control RNA with ChromHMM promoter states and transcribed states. Active transcription start site (TssA), flanking active TSS (TssAFlnk), strong (Tx) and weak (TxWk) transcription regions are shown. ( E ) Left: Overlap of TriplexRNA (DNA-IP), chromatin-associated RNA and nuclear RNA with different classes of repeat elements. Right: Abundance of simple repeat subclasses. ( F ) Abundance of TriplexRNA (DNA-IP) and control RNAs overlapping super-enhancers in <t>HeLa</t> S3 cells. Data are from HeLa S3 cells. Adjusted P -values
    Nebnext Poly A Mrna Magnetic Isolation Module, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 99/100, based on 295 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    New England Biolabs ultra ii directional rna library prep kit
    NEAT1 forms triplexes at numerous genomic sites. ( A ) NEAT1 profiles in TriplexRNA-seq (DNA-IP) (red) and nuclear <t>RNA</t> (blue) from HeLa S3 and U2OS cells with shaded TFR1 and TFR2. Minus (-) and plus (+) strands are shown. The position and sequence of NEAT1-TFR1 and -TFR2 are shown below. ( B ) EMSAs using 10 or 100 pmol of synthetic NEAT1 versions comprising TFR1 (40 or 52 nt) or TFR2 incubated with 0.25 pmol of double–stranded 32 P-labeled oligonucleotides which harbor sequences of NEAT1 target genes predicted from CHART-seq ( Supplementary Table S2 ). Reactions marked with an asterisk (*) were treated with 0.5 U RNase H. As a control, RNA without a putative TFR was used. Potential Hoogsteen base pairing between motifs and respective TFR sequences are shown; mismatches are marked (*). ( C ) Schematic depiction of the TFR-based capture assay. Biotinylated RNA <t>oligos</t> covering NEAT1-TFR1 and NEAT1-TFR2 were used to capture genomic DNA. ( D ) MEME motif analysis identifying consensus motifs in DNA captured by NEAT1-TFR1 (399 of top 500 peaks) and by NEAT1-TFR2 (500 of top 500 peaks ranked by peak P -value). Potential Hoogsteen base pairing between motifs and respective TFR sequences are shown; mismatches are marked (*). ( E ) TDF analysis of the triplex-forming potential of NEAT1-TFR1 and NEAT1-TFR2 RNAs with top 500 TFR-associated and control DNA peaks (ranked by peak P -value) compared to 500 randomized regions ( N = 1000, colored grey). P -values were obtained from one-tailed Mann–Whitney test. ( F ) Scheme presenting antisense oligo (ASO)-based capture of NEAT1-associated DNA. ( G ) Consensus motif in NEAT1-associated DNA sites (314 of top 500 peaks ranked by peak P -value). ( H ) TDF analysis predicting the triplex-forming potential of NEAT1 on ASO-captured DNA regions. Significant TFRs along NEAT1 are shown in orange, the number of target sites (DBS) for each TFR in purple. For TFR- and ASO-based capture assays nucleic acids isolated from HeLa S3 chromatin were used.
    Ultra Ii Directional Rna Library Prep Kit, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 99/100, based on 401 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    New England Biolabs quick t4 dna ligase
    NEAT1 forms triplexes at numerous genomic sites. ( A ) NEAT1 profiles in TriplexRNA-seq (DNA-IP) (red) and nuclear <t>RNA</t> (blue) from HeLa S3 and U2OS cells with shaded TFR1 and TFR2. Minus (-) and plus (+) strands are shown. The position and sequence of NEAT1-TFR1 and -TFR2 are shown below. ( B ) EMSAs using 10 or 100 pmol of synthetic NEAT1 versions comprising TFR1 (40 or 52 nt) or TFR2 incubated with 0.25 pmol of double–stranded 32 P-labeled oligonucleotides which harbor sequences of NEAT1 target genes predicted from CHART-seq ( Supplementary Table S2 ). Reactions marked with an asterisk (*) were treated with 0.5 U RNase H. As a control, RNA without a putative TFR was used. Potential Hoogsteen base pairing between motifs and respective TFR sequences are shown; mismatches are marked (*). ( C ) Schematic depiction of the TFR-based capture assay. Biotinylated RNA <t>oligos</t> covering NEAT1-TFR1 and NEAT1-TFR2 were used to capture genomic DNA. ( D ) MEME motif analysis identifying consensus motifs in DNA captured by NEAT1-TFR1 (399 of top 500 peaks) and by NEAT1-TFR2 (500 of top 500 peaks ranked by peak P -value). Potential Hoogsteen base pairing between motifs and respective TFR sequences are shown; mismatches are marked (*). ( E ) TDF analysis of the triplex-forming potential of NEAT1-TFR1 and NEAT1-TFR2 RNAs with top 500 TFR-associated and control DNA peaks (ranked by peak P -value) compared to 500 randomized regions ( N = 1000, colored grey). P -values were obtained from one-tailed Mann–Whitney test. ( F ) Scheme presenting antisense oligo (ASO)-based capture of NEAT1-associated DNA. ( G ) Consensus motif in NEAT1-associated DNA sites (314 of top 500 peaks ranked by peak P -value). ( H ) TDF analysis predicting the triplex-forming potential of NEAT1 on ASO-captured DNA regions. Significant TFRs along NEAT1 are shown in orange, the number of target sites (DBS) for each TFR in purple. For TFR- and ASO-based capture assays nucleic acids isolated from HeLa S3 chromatin were used.
    Quick T4 Dna Ligase, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 99/100, based on 657 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    NEAT1 forms triplexes at numerous genomic sites. ( A ) NEAT1 profiles in TriplexRNA-seq (DNA-IP) (red) and nuclear <t>RNA</t> (blue) from HeLa S3 and U2OS cells with shaded TFR1 and TFR2. Minus (-) and plus (+) strands are shown. The position and sequence of NEAT1-TFR1 and -TFR2 are shown below. ( B ) EMSAs using 10 or 100 pmol of synthetic NEAT1 versions comprising TFR1 (40 or 52 nt) or TFR2 incubated with 0.25 pmol of double–stranded 32 P-labeled oligonucleotides which harbor sequences of NEAT1 target genes predicted from CHART-seq ( Supplementary Table S2 ). Reactions marked with an asterisk (*) were treated with 0.5 U RNase H. As a control, RNA without a putative TFR was used. Potential Hoogsteen base pairing between motifs and respective TFR sequences are shown; mismatches are marked (*). ( C ) Schematic depiction of the TFR-based capture assay. Biotinylated RNA <t>oligos</t> covering NEAT1-TFR1 and NEAT1-TFR2 were used to capture genomic DNA. ( D ) MEME motif analysis identifying consensus motifs in DNA captured by NEAT1-TFR1 (399 of top 500 peaks) and by NEAT1-TFR2 (500 of top 500 peaks ranked by peak P -value). Potential Hoogsteen base pairing between motifs and respective TFR sequences are shown; mismatches are marked (*). ( E ) TDF analysis of the triplex-forming potential of NEAT1-TFR1 and NEAT1-TFR2 RNAs with top 500 TFR-associated and control DNA peaks (ranked by peak P -value) compared to 500 randomized regions ( N = 1000, colored grey). P -values were obtained from one-tailed Mann–Whitney test. ( F ) Scheme presenting antisense oligo (ASO)-based capture of NEAT1-associated DNA. ( G ) Consensus motif in NEAT1-associated DNA sites (314 of top 500 peaks ranked by peak P -value). ( H ) TDF analysis predicting the triplex-forming potential of NEAT1 on ASO-captured DNA regions. Significant TFRs along NEAT1 are shown in orange, the number of target sites (DBS) for each TFR in purple. For TFR- and ASO-based capture assays nucleic acids isolated from HeLa S3 chromatin were used.
    Ultra Rna Ii Library Prep Kit, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 99/100, based on 11 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    SIRT7 is involved in pre-rRNA processing. ( a ) Knockdown of SIRT7 impairs pre-rRNA synthesis and processing in vivo . U2OS cells transfected with control (siCtrl) or SIRT7-specific siRNAs (siSIRT7) were metabolically labelled with 3 H-uridine. <t>RNA</t> was analysed by agarose gel electrophoresis and fluorography. The bar diagram shows quantification of the processing intermediates, values from siCtrl cells being set to 1. ( b ) In vitro processing assay. Extracts from L1210 cells were incubated with 32 P-labelled RNA comprising the 5′ETS depicted in the scheme above. 32 P-labelled RNA and cleavage products were analysed by gel electrophoresis and PhosphorImaging. See also Supplementary Fig. 3a . ( c ) 5′ETS processing is inhibited by NAM. The assay contained radiolabelled RNA (+541/+1290) and extracts from L1210 cells cultured for 6 h in the absence or presence of NAM. ( d ) Processing is enhanced by NAD + . Processing assays containing radiolabelled RNA (+541/+1290) were substituted with NAD + as indicated. ( e ) The catalytic activity of SIRT7 is required for pre-rRNA cleavage. Assays were supplemented with 15 or 30 ng of purified wildtype (WT) or mutant (H187Y) Flag-SIRT7 ( Supplementary Fig. 3b ). ( f ) Depletion of SIRT7 impairs processing. SIRT7 was depleted from L1210 cells by shRNAs (shSIRT7-1, shSIRT7-2, Supplementary Fig. 3c ). Extracts from non-infected cells (−) or cells expressing control shRNA (shCtrl) served as control (left). To rescue impaired cleavage, 15 ng of wild-type Flag-SIRT7 (WT) or mutant H187Y (HY) were added to SIRT7-depleted extracts (right). ( g ) Depletion of U3 snoRNA abolishes processing. U3 snoRNA was depleted by preincubating extracts with U3-specific antisense <t>oligos</t> (ASO, 50 ng μl −1 ) and 2 U of RNase H ( Supplementary Fig. 3d ). In vitro processing was performed with undepleted (−) or depleted extracts in the absence or presence of 15 ng Flag-SIRT7. Bar diagrams in c – g show quantification of the ratio of cleaved versus uncleaved transcripts, presented as mean±s.d. from three independent experiments (* P
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    Image Search Results


    Identification and global characterization of DNA-associated RNA. ( A ) Scatter plots showing the correlation between TriplexRNA isolated by DNA-IP and SPRI selection (left) and TriplexRNA (DNA-IP) and control RNA (middle, right). Pearson correlation coefficient ( R ) across 7148 genes overlapping peaks is shown. Green diagonal line x = y . Some representative genes that overlap TriplexRNAs and control RNAs are highlighted. ( B ) Pie charts depicting the genomic distribution of TriplexRNA (DNA-IP) compared to chromatin-associated and nuclear RNA peaks, excluding intronic and exonic gene regions. Upstream and downstream regions are defined within 2.5 kb proximity of the closest gene. ( C ) Pie chart showing classification of long noncoding RNAs that overlap TriplexRNA (DNA-IP), chromatin-associated and nuclear RNA. ( D ) Association of TriplexRNA (DNA-IP) and control RNA with ChromHMM promoter states and transcribed states. Active transcription start site (TssA), flanking active TSS (TssAFlnk), strong (Tx) and weak (TxWk) transcription regions are shown. ( E ) Left: Overlap of TriplexRNA (DNA-IP), chromatin-associated RNA and nuclear RNA with different classes of repeat elements. Right: Abundance of simple repeat subclasses. ( F ) Abundance of TriplexRNA (DNA-IP) and control RNAs overlapping super-enhancers in HeLa S3 cells. Data are from HeLa S3 cells. Adjusted P -values

    Journal: Nucleic Acids Research

    Article Title: Isolation and genome-wide characterization of cellular DNA:RNA triplex structures

    doi: 10.1093/nar/gky1305

    Figure Lengend Snippet: Identification and global characterization of DNA-associated RNA. ( A ) Scatter plots showing the correlation between TriplexRNA isolated by DNA-IP and SPRI selection (left) and TriplexRNA (DNA-IP) and control RNA (middle, right). Pearson correlation coefficient ( R ) across 7148 genes overlapping peaks is shown. Green diagonal line x = y . Some representative genes that overlap TriplexRNAs and control RNAs are highlighted. ( B ) Pie charts depicting the genomic distribution of TriplexRNA (DNA-IP) compared to chromatin-associated and nuclear RNA peaks, excluding intronic and exonic gene regions. Upstream and downstream regions are defined within 2.5 kb proximity of the closest gene. ( C ) Pie chart showing classification of long noncoding RNAs that overlap TriplexRNA (DNA-IP), chromatin-associated and nuclear RNA. ( D ) Association of TriplexRNA (DNA-IP) and control RNA with ChromHMM promoter states and transcribed states. Active transcription start site (TssA), flanking active TSS (TssAFlnk), strong (Tx) and weak (TxWk) transcription regions are shown. ( E ) Left: Overlap of TriplexRNA (DNA-IP), chromatin-associated RNA and nuclear RNA with different classes of repeat elements. Right: Abundance of simple repeat subclasses. ( F ) Abundance of TriplexRNA (DNA-IP) and control RNAs overlapping super-enhancers in HeLa S3 cells. Data are from HeLa S3 cells. Adjusted P -values

    Article Snippet: DNA libraries were prepared from at least three independent experiments from HeLa S3 cells using the NEBNext Ultra II DNA Library Prep Kit for Illumina (NEB) and NEBNext Multiplex Oligos for Illumina (NEB).

    Techniques: Isolation, Selection

    NEAT1 forms triplexes at numerous genomic sites. ( A ) NEAT1 profiles in TriplexRNA-seq (DNA-IP) (red) and nuclear RNA (blue) from HeLa S3 and U2OS cells with shaded TFR1 and TFR2. Minus (-) and plus (+) strands are shown. The position and sequence of NEAT1-TFR1 and -TFR2 are shown below. ( B ) EMSAs using 10 or 100 pmol of synthetic NEAT1 versions comprising TFR1 (40 or 52 nt) or TFR2 incubated with 0.25 pmol of double–stranded 32 P-labeled oligonucleotides which harbor sequences of NEAT1 target genes predicted from CHART-seq ( Supplementary Table S2 ). Reactions marked with an asterisk (*) were treated with 0.5 U RNase H. As a control, RNA without a putative TFR was used. Potential Hoogsteen base pairing between motifs and respective TFR sequences are shown; mismatches are marked (*). ( C ) Schematic depiction of the TFR-based capture assay. Biotinylated RNA oligos covering NEAT1-TFR1 and NEAT1-TFR2 were used to capture genomic DNA. ( D ) MEME motif analysis identifying consensus motifs in DNA captured by NEAT1-TFR1 (399 of top 500 peaks) and by NEAT1-TFR2 (500 of top 500 peaks ranked by peak P -value). Potential Hoogsteen base pairing between motifs and respective TFR sequences are shown; mismatches are marked (*). ( E ) TDF analysis of the triplex-forming potential of NEAT1-TFR1 and NEAT1-TFR2 RNAs with top 500 TFR-associated and control DNA peaks (ranked by peak P -value) compared to 500 randomized regions ( N = 1000, colored grey). P -values were obtained from one-tailed Mann–Whitney test. ( F ) Scheme presenting antisense oligo (ASO)-based capture of NEAT1-associated DNA. ( G ) Consensus motif in NEAT1-associated DNA sites (314 of top 500 peaks ranked by peak P -value). ( H ) TDF analysis predicting the triplex-forming potential of NEAT1 on ASO-captured DNA regions. Significant TFRs along NEAT1 are shown in orange, the number of target sites (DBS) for each TFR in purple. For TFR- and ASO-based capture assays nucleic acids isolated from HeLa S3 chromatin were used.

    Journal: Nucleic Acids Research

    Article Title: Isolation and genome-wide characterization of cellular DNA:RNA triplex structures

    doi: 10.1093/nar/gky1305

    Figure Lengend Snippet: NEAT1 forms triplexes at numerous genomic sites. ( A ) NEAT1 profiles in TriplexRNA-seq (DNA-IP) (red) and nuclear RNA (blue) from HeLa S3 and U2OS cells with shaded TFR1 and TFR2. Minus (-) and plus (+) strands are shown. The position and sequence of NEAT1-TFR1 and -TFR2 are shown below. ( B ) EMSAs using 10 or 100 pmol of synthetic NEAT1 versions comprising TFR1 (40 or 52 nt) or TFR2 incubated with 0.25 pmol of double–stranded 32 P-labeled oligonucleotides which harbor sequences of NEAT1 target genes predicted from CHART-seq ( Supplementary Table S2 ). Reactions marked with an asterisk (*) were treated with 0.5 U RNase H. As a control, RNA without a putative TFR was used. Potential Hoogsteen base pairing between motifs and respective TFR sequences are shown; mismatches are marked (*). ( C ) Schematic depiction of the TFR-based capture assay. Biotinylated RNA oligos covering NEAT1-TFR1 and NEAT1-TFR2 were used to capture genomic DNA. ( D ) MEME motif analysis identifying consensus motifs in DNA captured by NEAT1-TFR1 (399 of top 500 peaks) and by NEAT1-TFR2 (500 of top 500 peaks ranked by peak P -value). Potential Hoogsteen base pairing between motifs and respective TFR sequences are shown; mismatches are marked (*). ( E ) TDF analysis of the triplex-forming potential of NEAT1-TFR1 and NEAT1-TFR2 RNAs with top 500 TFR-associated and control DNA peaks (ranked by peak P -value) compared to 500 randomized regions ( N = 1000, colored grey). P -values were obtained from one-tailed Mann–Whitney test. ( F ) Scheme presenting antisense oligo (ASO)-based capture of NEAT1-associated DNA. ( G ) Consensus motif in NEAT1-associated DNA sites (314 of top 500 peaks ranked by peak P -value). ( H ) TDF analysis predicting the triplex-forming potential of NEAT1 on ASO-captured DNA regions. Significant TFRs along NEAT1 are shown in orange, the number of target sites (DBS) for each TFR in purple. For TFR- and ASO-based capture assays nucleic acids isolated from HeLa S3 chromatin were used.

    Article Snippet: DNA libraries were prepared from at least three independent experiments from HeLa S3 cells using the NEBNext Ultra II DNA Library Prep Kit for Illumina (NEB) and NEBNext Multiplex Oligos for Illumina (NEB).

    Techniques: Sequencing, Incubation, Labeling, One-tailed Test, MANN-WHITNEY, Allele-specific Oligonucleotide, Isolation

    Enrichment of triplex-forming RNAs. ( A ) Schematic overview of the method to enrich DNA-associated RNA. ( B ) RT-qPCR monitoring the indicated RNAs recovered from HeLa S3 cells, nuclei and purified chromatin. Values are normalized to cellular RNA (±SEM, N = 3). ( C ) Polyacrylamide gel electrophoresis of 5′-labeled RNA enriched by SPRI-size selection. Control samples were treated with DNase I before size selection or with RNase A before gel loading. ( D ) RT-qPCR analysis of DNA-associated RNA from HeLa S3 cells isolated by SPRI-size selection. Values are normalized to cellular RNA. Control samples were treated with DNase I before size selection (±SEM, N = 3). ( E ) RNA-seq profiles for KHPS1 in DNA-associated RNAs (DNA-IP) and nuclear RNA from U2OS cells. The overlap with the TFR of KHPS1 is shaded. Minus (–) and plus (+) strands are shown.

    Journal: Nucleic Acids Research

    Article Title: Isolation and genome-wide characterization of cellular DNA:RNA triplex structures

    doi: 10.1093/nar/gky1305

    Figure Lengend Snippet: Enrichment of triplex-forming RNAs. ( A ) Schematic overview of the method to enrich DNA-associated RNA. ( B ) RT-qPCR monitoring the indicated RNAs recovered from HeLa S3 cells, nuclei and purified chromatin. Values are normalized to cellular RNA (±SEM, N = 3). ( C ) Polyacrylamide gel electrophoresis of 5′-labeled RNA enriched by SPRI-size selection. Control samples were treated with DNase I before size selection or with RNase A before gel loading. ( D ) RT-qPCR analysis of DNA-associated RNA from HeLa S3 cells isolated by SPRI-size selection. Values are normalized to cellular RNA. Control samples were treated with DNase I before size selection (±SEM, N = 3). ( E ) RNA-seq profiles for KHPS1 in DNA-associated RNAs (DNA-IP) and nuclear RNA from U2OS cells. The overlap with the TFR of KHPS1 is shaded. Minus (–) and plus (+) strands are shown.

    Article Snippet: DNA libraries were prepared from at least three independent experiments from HeLa S3 cells using the NEBNext Ultra II DNA Library Prep Kit for Illumina (NEB) and NEBNext Multiplex Oligos for Illumina (NEB).

    Techniques: Quantitative RT-PCR, Purification, Polyacrylamide Gel Electrophoresis, Labeling, Selection, Isolation, RNA Sequencing Assay

    Isolation and identification of RNA-associated DNAs. ( A ) Scheme illustrating the method used to isolate RNA-associated DNA (TriplexDNA). ( B ) Pie chart depicting the genomic distribution of TriplexDNA peaks. Upstream and downstream regions are defined within 2.5 kb proximity of the closest gene. The bar diagrams at the right display the fold change in the distribution of the respective regions in TriplexDNA compared to control DNA. ( C ) Line plots depicting the mean values of TriplexDNA-seq signals over TSS and TTS of 890 genes that overlap RNA-associated DNA peaks. Interval defined by maximum and minimum values is shaded. ( D ) TriplexDNA-seq regions overlapping DNase Hypersensitive Sites (DNase HS) in HeLa S3 cells provided by ENCODE. ( E ) Abundance of TriplexDNA regions associated with the indicated ChromHMM states. Active transcription start site (TssA), flanking active TSS (TssAFlnk), strong and weak (Tx, TxWk) transcription, heterochromatin (Het) and Polycomb-repressed (RepPC) regions are shown. ( F ) Top: Overlap of TriplexDNA and control DNA with different classes of repeat elements. Bottom: Abundance of predominating repeat subclasses in TriplexDNA. (G) MEME motif analysis identifying purine-rich consensus motifs in randomly selected 500 TriplexDNA peaks. Data are from HeLa S3 cells. Adjusted P -values

    Journal: Nucleic Acids Research

    Article Title: Isolation and genome-wide characterization of cellular DNA:RNA triplex structures

    doi: 10.1093/nar/gky1305

    Figure Lengend Snippet: Isolation and identification of RNA-associated DNAs. ( A ) Scheme illustrating the method used to isolate RNA-associated DNA (TriplexDNA). ( B ) Pie chart depicting the genomic distribution of TriplexDNA peaks. Upstream and downstream regions are defined within 2.5 kb proximity of the closest gene. The bar diagrams at the right display the fold change in the distribution of the respective regions in TriplexDNA compared to control DNA. ( C ) Line plots depicting the mean values of TriplexDNA-seq signals over TSS and TTS of 890 genes that overlap RNA-associated DNA peaks. Interval defined by maximum and minimum values is shaded. ( D ) TriplexDNA-seq regions overlapping DNase Hypersensitive Sites (DNase HS) in HeLa S3 cells provided by ENCODE. ( E ) Abundance of TriplexDNA regions associated with the indicated ChromHMM states. Active transcription start site (TssA), flanking active TSS (TssAFlnk), strong and weak (Tx, TxWk) transcription, heterochromatin (Het) and Polycomb-repressed (RepPC) regions are shown. ( F ) Top: Overlap of TriplexDNA and control DNA with different classes of repeat elements. Bottom: Abundance of predominating repeat subclasses in TriplexDNA. (G) MEME motif analysis identifying purine-rich consensus motifs in randomly selected 500 TriplexDNA peaks. Data are from HeLa S3 cells. Adjusted P -values

    Article Snippet: DNA libraries were prepared from at least three independent experiments from HeLa S3 cells using the NEBNext Ultra II DNA Library Prep Kit for Illumina (NEB) and NEBNext Multiplex Oligos for Illumina (NEB).

    Techniques: Isolation

    Validation of triplex-forming RNA and DNAs. ( A ) TDF analysis predicting the potential of top 1000 enriched TriplexRNA (DNA-IP) regions (ranked by peak P -value) to bind to active promoters defined by ChromHMM. Number of TFRs in RNA (per kilobase of RNA, left) and the number of putative DBSs at promoters (per kilobase of RNA, right) are shown. Boxplot borders are defined by the 1st and 3rd quantiles of the distributions, the middle line corresponds to the median value. The top whisker denotes the maximum value within the third quartile plus 1.5 times the interquartile range (bottom whisker is defined analogously). Dark gray dots represent outliers with values higher or lower than whiskers. Further box plots are based on the same definitions. ( B ) Motif analysis of triplexes formed between TriplexRNA (DNA-IP) and active promoters. The diagram depicts the fraction of antiparallel and parallel triplexes with the respective motif and nucleotide composition of TFRs in TriplexRNA. ( C ) TDF analysis comparing the triplex-forming potential of top 2000 TriplexDNA-seq regions with top 1000 TriplexRNA (DNA-IP) (ranked by peak P -value). The number of putative DBSs (per kilobase of RNA) is shown. ( D ) Motif analysis of predicted triplexes formed between TriplexRNAs (DNA-IP) and TriplexDNA. The diagram depicts the fraction of antiparallel and parallel triplexes, with the respective motif and nucleotide composition of TFRs in TriplexRNA. ( E ) Box plot classifying triplex interactions between TriplexRNAs (DNA-IP) and TriplexDNA-seq regions as cis ( > 10 kb in the same chromosome) and trans (at different chromosomes) interactions, excluding underrepresented local interactions (within 10 kb distance). ( F ) EMSAs using 10 or 100 pmol of synthetic TriplexRNAs and 0.25 pmol of double–stranded 32 P-labeled oligonucleotides comprising target regions from TriplexDNA ( Supplementary Table S2 ). Reactions marked with an asterisk (*) were treated with 0.5 U RNase H. As a control (C), RNA without a putative TFR was used. Potential Hoogsteen base pairing between motifs and respective TFR sequences are shown; mismatches are marked (*). TriplexRNA-seq and TriplexDNA-seq data are from HeLa S3 cells. Adjusted P -values

    Journal: Nucleic Acids Research

    Article Title: Isolation and genome-wide characterization of cellular DNA:RNA triplex structures

    doi: 10.1093/nar/gky1305

    Figure Lengend Snippet: Validation of triplex-forming RNA and DNAs. ( A ) TDF analysis predicting the potential of top 1000 enriched TriplexRNA (DNA-IP) regions (ranked by peak P -value) to bind to active promoters defined by ChromHMM. Number of TFRs in RNA (per kilobase of RNA, left) and the number of putative DBSs at promoters (per kilobase of RNA, right) are shown. Boxplot borders are defined by the 1st and 3rd quantiles of the distributions, the middle line corresponds to the median value. The top whisker denotes the maximum value within the third quartile plus 1.5 times the interquartile range (bottom whisker is defined analogously). Dark gray dots represent outliers with values higher or lower than whiskers. Further box plots are based on the same definitions. ( B ) Motif analysis of triplexes formed between TriplexRNA (DNA-IP) and active promoters. The diagram depicts the fraction of antiparallel and parallel triplexes with the respective motif and nucleotide composition of TFRs in TriplexRNA. ( C ) TDF analysis comparing the triplex-forming potential of top 2000 TriplexDNA-seq regions with top 1000 TriplexRNA (DNA-IP) (ranked by peak P -value). The number of putative DBSs (per kilobase of RNA) is shown. ( D ) Motif analysis of predicted triplexes formed between TriplexRNAs (DNA-IP) and TriplexDNA. The diagram depicts the fraction of antiparallel and parallel triplexes, with the respective motif and nucleotide composition of TFRs in TriplexRNA. ( E ) Box plot classifying triplex interactions between TriplexRNAs (DNA-IP) and TriplexDNA-seq regions as cis ( > 10 kb in the same chromosome) and trans (at different chromosomes) interactions, excluding underrepresented local interactions (within 10 kb distance). ( F ) EMSAs using 10 or 100 pmol of synthetic TriplexRNAs and 0.25 pmol of double–stranded 32 P-labeled oligonucleotides comprising target regions from TriplexDNA ( Supplementary Table S2 ). Reactions marked with an asterisk (*) were treated with 0.5 U RNase H. As a control (C), RNA without a putative TFR was used. Potential Hoogsteen base pairing between motifs and respective TFR sequences are shown; mismatches are marked (*). TriplexRNA-seq and TriplexDNA-seq data are from HeLa S3 cells. Adjusted P -values

    Article Snippet: DNA libraries were prepared from at least three independent experiments from HeLa S3 cells using the NEBNext Ultra II DNA Library Prep Kit for Illumina (NEB) and NEBNext Multiplex Oligos for Illumina (NEB).

    Techniques: Whisker Assay, Labeling

    NEAT1 forms triplexes at numerous genomic sites. ( A ) NEAT1 profiles in TriplexRNA-seq (DNA-IP) (red) and nuclear RNA (blue) from HeLa S3 and U2OS cells with shaded TFR1 and TFR2. Minus (-) and plus (+) strands are shown. The position and sequence of NEAT1-TFR1 and -TFR2 are shown below. ( B ) EMSAs using 10 or 100 pmol of synthetic NEAT1 versions comprising TFR1 (40 or 52 nt) or TFR2 incubated with 0.25 pmol of double–stranded 32 P-labeled oligonucleotides which harbor sequences of NEAT1 target genes predicted from CHART-seq ( Supplementary Table S2 ). Reactions marked with an asterisk (*) were treated with 0.5 U RNase H. As a control, RNA without a putative TFR was used. Potential Hoogsteen base pairing between motifs and respective TFR sequences are shown; mismatches are marked (*). ( C ) Schematic depiction of the TFR-based capture assay. Biotinylated RNA oligos covering NEAT1-TFR1 and NEAT1-TFR2 were used to capture genomic DNA. ( D ) MEME motif analysis identifying consensus motifs in DNA captured by NEAT1-TFR1 (399 of top 500 peaks) and by NEAT1-TFR2 (500 of top 500 peaks ranked by peak P -value). Potential Hoogsteen base pairing between motifs and respective TFR sequences are shown; mismatches are marked (*). ( E ) TDF analysis of the triplex-forming potential of NEAT1-TFR1 and NEAT1-TFR2 RNAs with top 500 TFR-associated and control DNA peaks (ranked by peak P -value) compared to 500 randomized regions ( N = 1000, colored grey). P -values were obtained from one-tailed Mann–Whitney test. ( F ) Scheme presenting antisense oligo (ASO)-based capture of NEAT1-associated DNA. ( G ) Consensus motif in NEAT1-associated DNA sites (314 of top 500 peaks ranked by peak P -value). ( H ) TDF analysis predicting the triplex-forming potential of NEAT1 on ASO-captured DNA regions. Significant TFRs along NEAT1 are shown in orange, the number of target sites (DBS) for each TFR in purple. For TFR- and ASO-based capture assays nucleic acids isolated from HeLa S3 chromatin were used.

    Journal: Nucleic Acids Research

    Article Title: Isolation and genome-wide characterization of cellular DNA:RNA triplex structures

    doi: 10.1093/nar/gky1305

    Figure Lengend Snippet: NEAT1 forms triplexes at numerous genomic sites. ( A ) NEAT1 profiles in TriplexRNA-seq (DNA-IP) (red) and nuclear RNA (blue) from HeLa S3 and U2OS cells with shaded TFR1 and TFR2. Minus (-) and plus (+) strands are shown. The position and sequence of NEAT1-TFR1 and -TFR2 are shown below. ( B ) EMSAs using 10 or 100 pmol of synthetic NEAT1 versions comprising TFR1 (40 or 52 nt) or TFR2 incubated with 0.25 pmol of double–stranded 32 P-labeled oligonucleotides which harbor sequences of NEAT1 target genes predicted from CHART-seq ( Supplementary Table S2 ). Reactions marked with an asterisk (*) were treated with 0.5 U RNase H. As a control, RNA without a putative TFR was used. Potential Hoogsteen base pairing between motifs and respective TFR sequences are shown; mismatches are marked (*). ( C ) Schematic depiction of the TFR-based capture assay. Biotinylated RNA oligos covering NEAT1-TFR1 and NEAT1-TFR2 were used to capture genomic DNA. ( D ) MEME motif analysis identifying consensus motifs in DNA captured by NEAT1-TFR1 (399 of top 500 peaks) and by NEAT1-TFR2 (500 of top 500 peaks ranked by peak P -value). Potential Hoogsteen base pairing between motifs and respective TFR sequences are shown; mismatches are marked (*). ( E ) TDF analysis of the triplex-forming potential of NEAT1-TFR1 and NEAT1-TFR2 RNAs with top 500 TFR-associated and control DNA peaks (ranked by peak P -value) compared to 500 randomized regions ( N = 1000, colored grey). P -values were obtained from one-tailed Mann–Whitney test. ( F ) Scheme presenting antisense oligo (ASO)-based capture of NEAT1-associated DNA. ( G ) Consensus motif in NEAT1-associated DNA sites (314 of top 500 peaks ranked by peak P -value). ( H ) TDF analysis predicting the triplex-forming potential of NEAT1 on ASO-captured DNA regions. Significant TFRs along NEAT1 are shown in orange, the number of target sites (DBS) for each TFR in purple. For TFR- and ASO-based capture assays nucleic acids isolated from HeLa S3 chromatin were used.

    Article Snippet: Libraries were prepared using the NEBNext Ultra II Directional RNA Library Prep Kit and NEBNext Multiplex Oligos for Illumina (NEB).

    Techniques: Sequencing, Incubation, Labeling, One-tailed Test, MANN-WHITNEY, Allele-specific Oligonucleotide, Isolation

    Validation of triplex-forming RNA and DNAs. ( A ) TDF analysis predicting the potential of top 1000 enriched TriplexRNA (DNA-IP) regions (ranked by peak P -value) to bind to active promoters defined by ChromHMM. Number of TFRs in RNA (per kilobase of RNA, left) and the number of putative DBSs at promoters (per kilobase of RNA, right) are shown. Boxplot borders are defined by the 1st and 3rd quantiles of the distributions, the middle line corresponds to the median value. The top whisker denotes the maximum value within the third quartile plus 1.5 times the interquartile range (bottom whisker is defined analogously). Dark gray dots represent outliers with values higher or lower than whiskers. Further box plots are based on the same definitions. ( B ) Motif analysis of triplexes formed between TriplexRNA (DNA-IP) and active promoters. The diagram depicts the fraction of antiparallel and parallel triplexes with the respective motif and nucleotide composition of TFRs in TriplexRNA. ( C ) TDF analysis comparing the triplex-forming potential of top 2000 TriplexDNA-seq regions with top 1000 TriplexRNA (DNA-IP) (ranked by peak P -value). The number of putative DBSs (per kilobase of RNA) is shown. ( D ) Motif analysis of predicted triplexes formed between TriplexRNAs (DNA-IP) and TriplexDNA. The diagram depicts the fraction of antiparallel and parallel triplexes, with the respective motif and nucleotide composition of TFRs in TriplexRNA. ( E ) Box plot classifying triplex interactions between TriplexRNAs (DNA-IP) and TriplexDNA-seq regions as cis ( > 10 kb in the same chromosome) and trans (at different chromosomes) interactions, excluding underrepresented local interactions (within 10 kb distance). ( F ) EMSAs using 10 or 100 pmol of synthetic TriplexRNAs and 0.25 pmol of double–stranded 32 P-labeled oligonucleotides comprising target regions from TriplexDNA ( Supplementary Table S2 ). Reactions marked with an asterisk (*) were treated with 0.5 U RNase H. As a control (C), RNA without a putative TFR was used. Potential Hoogsteen base pairing between motifs and respective TFR sequences are shown; mismatches are marked (*). TriplexRNA-seq and TriplexDNA-seq data are from HeLa S3 cells. Adjusted P -values

    Journal: Nucleic Acids Research

    Article Title: Isolation and genome-wide characterization of cellular DNA:RNA triplex structures

    doi: 10.1093/nar/gky1305

    Figure Lengend Snippet: Validation of triplex-forming RNA and DNAs. ( A ) TDF analysis predicting the potential of top 1000 enriched TriplexRNA (DNA-IP) regions (ranked by peak P -value) to bind to active promoters defined by ChromHMM. Number of TFRs in RNA (per kilobase of RNA, left) and the number of putative DBSs at promoters (per kilobase of RNA, right) are shown. Boxplot borders are defined by the 1st and 3rd quantiles of the distributions, the middle line corresponds to the median value. The top whisker denotes the maximum value within the third quartile plus 1.5 times the interquartile range (bottom whisker is defined analogously). Dark gray dots represent outliers with values higher or lower than whiskers. Further box plots are based on the same definitions. ( B ) Motif analysis of triplexes formed between TriplexRNA (DNA-IP) and active promoters. The diagram depicts the fraction of antiparallel and parallel triplexes with the respective motif and nucleotide composition of TFRs in TriplexRNA. ( C ) TDF analysis comparing the triplex-forming potential of top 2000 TriplexDNA-seq regions with top 1000 TriplexRNA (DNA-IP) (ranked by peak P -value). The number of putative DBSs (per kilobase of RNA) is shown. ( D ) Motif analysis of predicted triplexes formed between TriplexRNAs (DNA-IP) and TriplexDNA. The diagram depicts the fraction of antiparallel and parallel triplexes, with the respective motif and nucleotide composition of TFRs in TriplexRNA. ( E ) Box plot classifying triplex interactions between TriplexRNAs (DNA-IP) and TriplexDNA-seq regions as cis ( > 10 kb in the same chromosome) and trans (at different chromosomes) interactions, excluding underrepresented local interactions (within 10 kb distance). ( F ) EMSAs using 10 or 100 pmol of synthetic TriplexRNAs and 0.25 pmol of double–stranded 32 P-labeled oligonucleotides comprising target regions from TriplexDNA ( Supplementary Table S2 ). Reactions marked with an asterisk (*) were treated with 0.5 U RNase H. As a control (C), RNA without a putative TFR was used. Potential Hoogsteen base pairing between motifs and respective TFR sequences are shown; mismatches are marked (*). TriplexRNA-seq and TriplexDNA-seq data are from HeLa S3 cells. Adjusted P -values

    Article Snippet: Libraries were prepared using the NEBNext Ultra II Directional RNA Library Prep Kit and NEBNext Multiplex Oligos for Illumina (NEB).

    Techniques: Whisker Assay, Labeling

    SIRT7 is involved in pre-rRNA processing. ( a ) Knockdown of SIRT7 impairs pre-rRNA synthesis and processing in vivo . U2OS cells transfected with control (siCtrl) or SIRT7-specific siRNAs (siSIRT7) were metabolically labelled with 3 H-uridine. RNA was analysed by agarose gel electrophoresis and fluorography. The bar diagram shows quantification of the processing intermediates, values from siCtrl cells being set to 1. ( b ) In vitro processing assay. Extracts from L1210 cells were incubated with 32 P-labelled RNA comprising the 5′ETS depicted in the scheme above. 32 P-labelled RNA and cleavage products were analysed by gel electrophoresis and PhosphorImaging. See also Supplementary Fig. 3a . ( c ) 5′ETS processing is inhibited by NAM. The assay contained radiolabelled RNA (+541/+1290) and extracts from L1210 cells cultured for 6 h in the absence or presence of NAM. ( d ) Processing is enhanced by NAD + . Processing assays containing radiolabelled RNA (+541/+1290) were substituted with NAD + as indicated. ( e ) The catalytic activity of SIRT7 is required for pre-rRNA cleavage. Assays were supplemented with 15 or 30 ng of purified wildtype (WT) or mutant (H187Y) Flag-SIRT7 ( Supplementary Fig. 3b ). ( f ) Depletion of SIRT7 impairs processing. SIRT7 was depleted from L1210 cells by shRNAs (shSIRT7-1, shSIRT7-2, Supplementary Fig. 3c ). Extracts from non-infected cells (−) or cells expressing control shRNA (shCtrl) served as control (left). To rescue impaired cleavage, 15 ng of wild-type Flag-SIRT7 (WT) or mutant H187Y (HY) were added to SIRT7-depleted extracts (right). ( g ) Depletion of U3 snoRNA abolishes processing. U3 snoRNA was depleted by preincubating extracts with U3-specific antisense oligos (ASO, 50 ng μl −1 ) and 2 U of RNase H ( Supplementary Fig. 3d ). In vitro processing was performed with undepleted (−) or depleted extracts in the absence or presence of 15 ng Flag-SIRT7. Bar diagrams in c – g show quantification of the ratio of cleaved versus uncleaved transcripts, presented as mean±s.d. from three independent experiments (* P

    Journal: Nature Communications

    Article Title: SIRT7-dependent deacetylation of the U3-55k protein controls pre-rRNA processing

    doi: 10.1038/ncomms10734

    Figure Lengend Snippet: SIRT7 is involved in pre-rRNA processing. ( a ) Knockdown of SIRT7 impairs pre-rRNA synthesis and processing in vivo . U2OS cells transfected with control (siCtrl) or SIRT7-specific siRNAs (siSIRT7) were metabolically labelled with 3 H-uridine. RNA was analysed by agarose gel electrophoresis and fluorography. The bar diagram shows quantification of the processing intermediates, values from siCtrl cells being set to 1. ( b ) In vitro processing assay. Extracts from L1210 cells were incubated with 32 P-labelled RNA comprising the 5′ETS depicted in the scheme above. 32 P-labelled RNA and cleavage products were analysed by gel electrophoresis and PhosphorImaging. See also Supplementary Fig. 3a . ( c ) 5′ETS processing is inhibited by NAM. The assay contained radiolabelled RNA (+541/+1290) and extracts from L1210 cells cultured for 6 h in the absence or presence of NAM. ( d ) Processing is enhanced by NAD + . Processing assays containing radiolabelled RNA (+541/+1290) were substituted with NAD + as indicated. ( e ) The catalytic activity of SIRT7 is required for pre-rRNA cleavage. Assays were supplemented with 15 or 30 ng of purified wildtype (WT) or mutant (H187Y) Flag-SIRT7 ( Supplementary Fig. 3b ). ( f ) Depletion of SIRT7 impairs processing. SIRT7 was depleted from L1210 cells by shRNAs (shSIRT7-1, shSIRT7-2, Supplementary Fig. 3c ). Extracts from non-infected cells (−) or cells expressing control shRNA (shCtrl) served as control (left). To rescue impaired cleavage, 15 ng of wild-type Flag-SIRT7 (WT) or mutant H187Y (HY) were added to SIRT7-depleted extracts (right). ( g ) Depletion of U3 snoRNA abolishes processing. U3 snoRNA was depleted by preincubating extracts with U3-specific antisense oligos (ASO, 50 ng μl −1 ) and 2 U of RNase H ( Supplementary Fig. 3d ). In vitro processing was performed with undepleted (−) or depleted extracts in the absence or presence of 15 ng Flag-SIRT7. Bar diagrams in c – g show quantification of the ratio of cleaved versus uncleaved transcripts, presented as mean±s.d. from three independent experiments (* P

    Article Snippet: The RNA-seq libraries were created using the NEBNext Ultra RNA Library Prep kit for Illumina (E7530) with NEBNext Multiplex Oligos for Illumina (E7300).

    Techniques: In Vivo, Transfection, Metabolic Labelling, Agarose Gel Electrophoresis, In Vitro, Incubation, Nucleic Acid Electrophoresis, Cell Culture, Activity Assay, Purification, Mutagenesis, Infection, Expressing, shRNA, Allele-specific Oligonucleotide

    Pre-rRNA transcription and processing are attenuated under stress. ( a ) Northern blot of pre-rRNA and processing intermediates from HEK293T cells that were untreated, exposed to hyperosmotic stress for 90 min (hypertonic), or recovered to regular medium for 60 min (hypertonic rel.). Membranes were probed with 32 P-labelled antisense riboprobe specific to 47S pre-rRNA (5'ETS, top) or with ITS1 oligos hybridizing to pre-rRNA intermediates (middle panel). ( b ) Acetylation of U3-55k is increased on different cellular stress conditions. HEK293T cells expressing Flag-U3-55k were treated with actinomycin D (Act D, 0.1 μg ml −1 , 4 h), AICAR (0.5 mM, 12 h) or exposed to hypertonic stress. Acetylation of immunopurified Flag-U3-55k and equal loading was monitored on western blots using anti-pan-AcK and anti-Flag antibodies. ( c ) Cellular localization of SIRT7 and U3-55k on hyperosmotic stress. Images showing localization of GFP-U3-55k and SIRT7 in normal conditions and on exposure to hyperosmotic stress for 90 min. Nuclei were stained with Hoechst 33342. Scale bars, 10 μm. ( d ) Overexpression of SIRT7 alleviates processing defects on hypertonic stress. Northern blot of RNA from parental U2OS cells and from cells which stably express GFP-SIRT7 (U2OS-GFP-SIRT7) using 5′ETS and ITS1 probes as in a . ( e ) CLIP-RT–qPCR monitoring binding of Flag-U3-55k to pre-rRNA, U3 snoRNA and U2 snRNA in HEK293T cells cultured in normo-osmotic medium or exposed to hypertonic stress for 90 min. Precipitated RNA was analysed by RT–qPCR using the indicated primers. Bars represent the means±s.d. from three biological repeats (* P

    Journal: Nature Communications

    Article Title: SIRT7-dependent deacetylation of the U3-55k protein controls pre-rRNA processing

    doi: 10.1038/ncomms10734

    Figure Lengend Snippet: Pre-rRNA transcription and processing are attenuated under stress. ( a ) Northern blot of pre-rRNA and processing intermediates from HEK293T cells that were untreated, exposed to hyperosmotic stress for 90 min (hypertonic), or recovered to regular medium for 60 min (hypertonic rel.). Membranes were probed with 32 P-labelled antisense riboprobe specific to 47S pre-rRNA (5'ETS, top) or with ITS1 oligos hybridizing to pre-rRNA intermediates (middle panel). ( b ) Acetylation of U3-55k is increased on different cellular stress conditions. HEK293T cells expressing Flag-U3-55k were treated with actinomycin D (Act D, 0.1 μg ml −1 , 4 h), AICAR (0.5 mM, 12 h) or exposed to hypertonic stress. Acetylation of immunopurified Flag-U3-55k and equal loading was monitored on western blots using anti-pan-AcK and anti-Flag antibodies. ( c ) Cellular localization of SIRT7 and U3-55k on hyperosmotic stress. Images showing localization of GFP-U3-55k and SIRT7 in normal conditions and on exposure to hyperosmotic stress for 90 min. Nuclei were stained with Hoechst 33342. Scale bars, 10 μm. ( d ) Overexpression of SIRT7 alleviates processing defects on hypertonic stress. Northern blot of RNA from parental U2OS cells and from cells which stably express GFP-SIRT7 (U2OS-GFP-SIRT7) using 5′ETS and ITS1 probes as in a . ( e ) CLIP-RT–qPCR monitoring binding of Flag-U3-55k to pre-rRNA, U3 snoRNA and U2 snRNA in HEK293T cells cultured in normo-osmotic medium or exposed to hypertonic stress for 90 min. Precipitated RNA was analysed by RT–qPCR using the indicated primers. Bars represent the means±s.d. from three biological repeats (* P

    Article Snippet: The RNA-seq libraries were created using the NEBNext Ultra RNA Library Prep kit for Illumina (E7530) with NEBNext Multiplex Oligos for Illumina (E7300).

    Techniques: Northern Blot, Expressing, Activated Clotting Time Assay, Western Blot, Staining, Over Expression, Stable Transfection, Cross-linking Immunoprecipitation, Quantitative RT-PCR, Binding Assay, Cell Culture