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  • 99
    New England Biolabs multiplex oligonucleotides
    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
    Multiplex Oligonucleotides, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 99/100, based on 212 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    New England Biolabs universal pcr primers
    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 <t>PCR</t> 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 <t>oligos</t> 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.
    Universal Pcr Primers, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 99/100, based on 62 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Image Search Results


    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

    SIRT7 is associated with pre-rRNA and snoRNAs. ( a ) SIRT7 CLIP-seq reads mapped to a custom annotation file of a human rDNA repeat (middle) or the transcribed region (bottom). The region encoding 18S, 5.8S and 28S rRNA is highlighted. SIRT7 reads after subtraction of IgG reads were normalized to input reads ( y axis). ( b ) Gene ontology categories of SIRT7 CLIP-seq peaks. The most representative clusters are shown according to the ajusted P value (−log 10 ). ( c ) SIRT7-bound snoRNAs comprise C/D box, H/ACA box snoRNAs and scaRNAs. The number ( n ) and relative abundance (%) of each snoRNA class associated with SIRT7 is presented. ( d ) U3, SNORA73A and 73B snoRNAs are overrepresented among SIRT7-associated snoRNAs. SIRT7 reads mapped to corresponding snoRNAs are indicated as percentage of all snoRNAs identified by CLIP-seq. ( e ) Comparison of SIRT7-associated RNAs under native and denaturing conditions. His/V5-tagged SIRT7 expressed in HEK293T cells was affinity-purified on Ni-NTA-agarose under native or denaturing conditions, and associated RNAs were detected by RT–qPCR. Lysates from non-transfected HEK293T cells were used for control (Ctrl). Associated pre-RNA was monitored by RT–qPCR using primer H1 ( Supplementary Table 3 ). Bars represent means±s.d. from three experiments. See also Supplementary Fig. 2b,d . ( f ) ChIP assays showing association of endogenous SIRT7 (left panel) or transiently overexpressed Flag-SIRT7 (right panel) with the indicated gene loci in HEK293T cells. rDNA was amplified using primers H4 (coding) and H18 (IGS; Supplementary Table 3 ). Bars represent means±s.d. from three experiments. See also Supplementary Fig. 2d .

    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 associated with pre-rRNA and snoRNAs. ( a ) SIRT7 CLIP-seq reads mapped to a custom annotation file of a human rDNA repeat (middle) or the transcribed region (bottom). The region encoding 18S, 5.8S and 28S rRNA is highlighted. SIRT7 reads after subtraction of IgG reads were normalized to input reads ( y axis). ( b ) Gene ontology categories of SIRT7 CLIP-seq peaks. The most representative clusters are shown according to the ajusted P value (−log 10 ). ( c ) SIRT7-bound snoRNAs comprise C/D box, H/ACA box snoRNAs and scaRNAs. The number ( n ) and relative abundance (%) of each snoRNA class associated with SIRT7 is presented. ( d ) U3, SNORA73A and 73B snoRNAs are overrepresented among SIRT7-associated snoRNAs. SIRT7 reads mapped to corresponding snoRNAs are indicated as percentage of all snoRNAs identified by CLIP-seq. ( e ) Comparison of SIRT7-associated RNAs under native and denaturing conditions. His/V5-tagged SIRT7 expressed in HEK293T cells was affinity-purified on Ni-NTA-agarose under native or denaturing conditions, and associated RNAs were detected by RT–qPCR. Lysates from non-transfected HEK293T cells were used for control (Ctrl). Associated pre-RNA was monitored by RT–qPCR using primer H1 ( Supplementary Table 3 ). Bars represent means±s.d. from three experiments. See also Supplementary Fig. 2b,d . ( f ) ChIP assays showing association of endogenous SIRT7 (left panel) or transiently overexpressed Flag-SIRT7 (right panel) with the indicated gene loci in HEK293T cells. rDNA was amplified using primers H4 (coding) and H18 (IGS; Supplementary Table 3 ). Bars represent means±s.d. from three experiments. See also Supplementary Fig. 2d .

    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: Cross-linking Immunoprecipitation, Affinity Purification, Quantitative RT-PCR, Transfection, Chromatin Immunoprecipitation, Amplification

    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

    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

    Detection of bisulfite-resistant cytosines in purified, linearized human mtDNA by bisulfite pyrosequencing using converted template-selective (A9515) and unselective (hND1) sequencing primers. Ratios of brCs were determined by bisulfite pyrosequencing (Mean±SD, triplicated assays). (A) The A9515 sequencing primer, which was highly selective to bisulfite-converted DNA, interrogated three CpG sites (CpG #3–5) whereas non-selective sequencing primer hND1 interrogated all these CpG sites plus two additional CpG sites (CpG #1 and 2). (B) Positive control assay was performed using in vitro partially methylated NCAs templates. High CpG methylation levels at three CpG sites (CpG #3–5) were detected using A9515 sequencing primer (CpG sites #1 and #2 were out of the assay coverage using this sequencing primer). hND1 sequencing primer detected high CpG methylation at all five CpG sites (CpG #1–5).

    Journal: PLoS ONE

    Article Title: Technical adequacy of bisulfite sequencing and pyrosequencing for detection of mitochondrial DNA methylation: Sources and avoidance of false-positive detection

    doi: 10.1371/journal.pone.0192722

    Figure Lengend Snippet: Detection of bisulfite-resistant cytosines in purified, linearized human mtDNA by bisulfite pyrosequencing using converted template-selective (A9515) and unselective (hND1) sequencing primers. Ratios of brCs were determined by bisulfite pyrosequencing (Mean±SD, triplicated assays). (A) The A9515 sequencing primer, which was highly selective to bisulfite-converted DNA, interrogated three CpG sites (CpG #3–5) whereas non-selective sequencing primer hND1 interrogated all these CpG sites plus two additional CpG sites (CpG #1 and 2). (B) Positive control assay was performed using in vitro partially methylated NCAs templates. High CpG methylation levels at three CpG sites (CpG #3–5) were detected using A9515 sequencing primer (CpG sites #1 and #2 were out of the assay coverage using this sequencing primer). hND1 sequencing primer detected high CpG methylation at all five CpG sites (CpG #1–5).

    Article Snippet: Equimolar mixture of shared mtDNA and lambda DNA was subjected to construction of Illumina shotgun bisulfite-seq deep sequencing libraries using NEBNEXT Ultra II DNA Library Prep Kit for Illumina, NEBNext Multiplex Oligos for Illumina (Methylated Adaptor, Index Primers Set 1), and the bisulfite conversion-compatible EpiMark Hot Start Taq DNA Polymerase (New England Biolabs).

    Techniques: Purification, Sequencing, Positive Control Assay, In Vitro, Methylation, CpG Methylation Assay

    Effects of unconverted DNA on false-positive detection of CpG methylation. Mixtures of bisulfite-converted and unconverted NCAs were subjected to bisulfite pyrosequencing using sequencing primers A9515 or hND1. Each bar represents mean±SD of three independent analyses.

    Journal: PLoS ONE

    Article Title: Technical adequacy of bisulfite sequencing and pyrosequencing for detection of mitochondrial DNA methylation: Sources and avoidance of false-positive detection

    doi: 10.1371/journal.pone.0192722

    Figure Lengend Snippet: Effects of unconverted DNA on false-positive detection of CpG methylation. Mixtures of bisulfite-converted and unconverted NCAs were subjected to bisulfite pyrosequencing using sequencing primers A9515 or hND1. Each bar represents mean±SD of three independent analyses.

    Article Snippet: Equimolar mixture of shared mtDNA and lambda DNA was subjected to construction of Illumina shotgun bisulfite-seq deep sequencing libraries using NEBNEXT Ultra II DNA Library Prep Kit for Illumina, NEBNext Multiplex Oligos for Illumina (Methylated Adaptor, Index Primers Set 1), and the bisulfite conversion-compatible EpiMark Hot Start Taq DNA Polymerase (New England Biolabs).

    Techniques: CpG Methylation Assay, Sequencing

    RepeatExplorer (RE) analysis of next-generation sequencing (NGS) data in Chenopodium diploids. ( A ) Cluster 61 of C. ficifolium demonstrate layouts that are typical for tandem repeats where nodes represent the sequence reads and edges between the nodes correspond to similarity hits; ( B ) Self-to-self comparisons of the contig 25 cluster 61 displayed as dot plots (genomic similarity search tool YASS program output) where parallel lines indicate tandem repeats (the distance between the diagonals equals the lengths of the motifs ~40 bp); ( C ) Agarose gel electrophoresis of PCR products obtained with primers designed from consensus monomer sequence of C. ficifolium (Cluster 61) showing typical ladder structure of tandem array.

    Journal: International Journal of Molecular Sciences

    Article Title: Natural History of a Satellite DNA Family: From the Ancestral Genome Component to Species-Specific Sequences, Concerted and Non-Concerted Evolution

    doi: 10.3390/ijms20051201

    Figure Lengend Snippet: RepeatExplorer (RE) analysis of next-generation sequencing (NGS) data in Chenopodium diploids. ( A ) Cluster 61 of C. ficifolium demonstrate layouts that are typical for tandem repeats where nodes represent the sequence reads and edges between the nodes correspond to similarity hits; ( B ) Self-to-self comparisons of the contig 25 cluster 61 displayed as dot plots (genomic similarity search tool YASS program output) where parallel lines indicate tandem repeats (the distance between the diagonals equals the lengths of the motifs ~40 bp); ( C ) Agarose gel electrophoresis of PCR products obtained with primers designed from consensus monomer sequence of C. ficifolium (Cluster 61) showing typical ladder structure of tandem array.

    Article Snippet: The individual libraries (corresponding to individual species) were enriched and indexed by unique barcodes using PCR with NEBNext Q5 HotStart HiFi PCR Master Mix and NEBNext Multiplex Oligos for Illumina (New England BioLabs) according to the manufacturer’s instructions.

    Techniques: Next-Generation Sequencing, Sequencing, Agarose Gel Electrophoresis, Polymerase Chain Reaction

    Agarose gel electrophoresis of PCR products obtained with primers designed from consensus monomer sequence of proposed high order repeat (HOR) units for determination of their physical counterparts. Cloned DNA fragments are shown by asterisks. The far-right line is an example of negative amplification of a computer-generated proposed HOR unit.

    Journal: International Journal of Molecular Sciences

    Article Title: Natural History of a Satellite DNA Family: From the Ancestral Genome Component to Species-Specific Sequences, Concerted and Non-Concerted Evolution

    doi: 10.3390/ijms20051201

    Figure Lengend Snippet: Agarose gel electrophoresis of PCR products obtained with primers designed from consensus monomer sequence of proposed high order repeat (HOR) units for determination of their physical counterparts. Cloned DNA fragments are shown by asterisks. The far-right line is an example of negative amplification of a computer-generated proposed HOR unit.

    Article Snippet: The individual libraries (corresponding to individual species) were enriched and indexed by unique barcodes using PCR with NEBNext Q5 HotStart HiFi PCR Master Mix and NEBNext Multiplex Oligos for Illumina (New England BioLabs) according to the manufacturer’s instructions.

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

    DDX1 Binds to G4 Structures in Intronic Switch RNAs (A) RNA oligonucleotides consisting of 4 tandem Sμ repeats (Sμ4G) or a G-to-C mutant (Sμ4Gmut). (B and C) RNA pull-down assays with protein extracts from (B) AID FLAG-HA or (C) AID KO CH12 cells, CIT stimulated for 48 hr. Western blots were analyzed for DDX1 and AID (FLAG tag) and RNA recovered from beads measured by dot blot. Representative results from at least 3 independent pull-downs. (D) Native electrophoretic mobility shift assays (EMSA) with 32 P-labeled Sμ4G and Sμ4Gmut RNA oligonucleotides and rDDX1 (WT) or rDDX1-K52A (ATPase mutant) proteins (1, 2, or 4 μg). Representative results from at least 3 independent assays. (E–H) CH12 cells were transfected with a pcDNA3 vector expressing GFP or N-terminal GFP-tagged human DDX1-K52A cDNA (GFP::DDX1-K52A), and cultured in UNS or CIT-stimulated conditions. (E) Percentage of GFP + cells 24 hr and 40 hr after transfection measured by flow cytometry (n = 4, mean ± SD). (F) Western blot of GFP + , fluorescence-activated cell sorted cells for DDX1 and Tubulin loading control (24 hr after transfection, 2 replicates). (G) Quantification of CSR in GFP – and GFP + -gated cell populations (40 hr after transfection; n = 4, mean ± SD). (H) DIP analyses with S9.6 antibody (IP) or no antibody control (–), 24 hr after transfection in CIT-stimulated conditions using Sα region probe 9. Values were normalized to probe 2 in each sample and probe 9 in shCtrl CIT cells in each experiment (n = 3, mean ± SD). See also Figure S4 .

    Journal: Molecular Cell

    Article Title: RNA Helicase DDX1 Converts RNA G-Quadruplex Structures into R-Loops to Promote IgH Class Switch Recombination

    doi: 10.1016/j.molcel.2018.04.001

    Figure Lengend Snippet: DDX1 Binds to G4 Structures in Intronic Switch RNAs (A) RNA oligonucleotides consisting of 4 tandem Sμ repeats (Sμ4G) or a G-to-C mutant (Sμ4Gmut). (B and C) RNA pull-down assays with protein extracts from (B) AID FLAG-HA or (C) AID KO CH12 cells, CIT stimulated for 48 hr. Western blots were analyzed for DDX1 and AID (FLAG tag) and RNA recovered from beads measured by dot blot. Representative results from at least 3 independent pull-downs. (D) Native electrophoretic mobility shift assays (EMSA) with 32 P-labeled Sμ4G and Sμ4Gmut RNA oligonucleotides and rDDX1 (WT) or rDDX1-K52A (ATPase mutant) proteins (1, 2, or 4 μg). Representative results from at least 3 independent assays. (E–H) CH12 cells were transfected with a pcDNA3 vector expressing GFP or N-terminal GFP-tagged human DDX1-K52A cDNA (GFP::DDX1-K52A), and cultured in UNS or CIT-stimulated conditions. (E) Percentage of GFP + cells 24 hr and 40 hr after transfection measured by flow cytometry (n = 4, mean ± SD). (F) Western blot of GFP + , fluorescence-activated cell sorted cells for DDX1 and Tubulin loading control (24 hr after transfection, 2 replicates). (G) Quantification of CSR in GFP – and GFP + -gated cell populations (40 hr after transfection; n = 4, mean ± SD). (H) DIP analyses with S9.6 antibody (IP) or no antibody control (–), 24 hr after transfection in CIT-stimulated conditions using Sα region probe 9. Values were normalized to probe 2 in each sample and probe 9 in shCtrl CIT cells in each experiment (n = 3, mean ± SD). See also Figure S4 .

    Article Snippet: Libraries were prepared from 100 ng RNA using the NEBNext Ultra Directional RNA Library Prep kit and NEBnext Multiplex Oligos (Index Primers Set 2) for Illumina according to manufacturer’s instructions (NEB).

    Techniques: Mutagenesis, Hemagglutination Assay, Gene Knockout, Western Blot, FLAG-tag, Dot Blot, Electrophoretic Mobility Shift Assay, Labeling, Transfection, Plasmid Preparation, Expressing, Cell Culture, Flow Cytometry, Cytometry, Fluorescence

    The newly developed mTSS-Capture method combined with paired-end sequencing maps genome-wide nucleosome distribution in primary patient samples and identifies bona fide nucleosome characteristics, concordant with other human nucleosome mapping studies A . Work-flow of the mTSS-seq method. Following MNase digestion using a titration of MNase, populations of mononucleosomally protected DNA and subnucleosomal fragments are isolated, and prepared as libraries for Illumina sequencing. Solution-based sequence capture is performed using biotinylated oligos, enabling the enrichment of fragments within 2kb of each transcription start site in the human genome. Paired-end 50bp sequencing was then performed on each index. B . Alignment of the mTSS-seq midpoints to the human genome using the UCSC genome browser for LAC patient #4137 Normal tissue is shown for chr11, hg19 ( http://genome.ucsc.edu ). Zooming in twice at 100X allows for further visualization of the sequence capture oligos surrounding the TSS in a 500kb and a 5kb region showing the ATM locus. C . Averaged, normalized reads per million (y-axis) from mTSS-seq plotted as fragments (gray) and midpoints (black), centered on and surrounding 2kb of the TSS for ~22,000 open reading frames in hg19 (x-axis). DNase I-hypersensitivity (GSM736580; green) and RNA polymerase II from ChIP-seq (GSM935299; blue) data from A549 cells are shown. (D) LAC patient 4137 Normal nucleosomal midpoints (blue track) were plotted in the UCSC genome browser against the published human lymphocyte nucleosome distribution maps by Gaffney et. al. (green track) for the ZNF451 and CCDC97 loci. Sequence capture oligos and corresponding RefSeq gene models are shown for each locus. Correlations are shown for ZNF451 and CCDC87, respectively.

    Journal: Oncotarget

    Article Title: Comprehensive nucleosome mapping of the human genome in cancer progression

    doi: 10.18632/oncotarget.6811

    Figure Lengend Snippet: The newly developed mTSS-Capture method combined with paired-end sequencing maps genome-wide nucleosome distribution in primary patient samples and identifies bona fide nucleosome characteristics, concordant with other human nucleosome mapping studies A . Work-flow of the mTSS-seq method. Following MNase digestion using a titration of MNase, populations of mononucleosomally protected DNA and subnucleosomal fragments are isolated, and prepared as libraries for Illumina sequencing. Solution-based sequence capture is performed using biotinylated oligos, enabling the enrichment of fragments within 2kb of each transcription start site in the human genome. Paired-end 50bp sequencing was then performed on each index. B . Alignment of the mTSS-seq midpoints to the human genome using the UCSC genome browser for LAC patient #4137 Normal tissue is shown for chr11, hg19 ( http://genome.ucsc.edu ). Zooming in twice at 100X allows for further visualization of the sequence capture oligos surrounding the TSS in a 500kb and a 5kb region showing the ATM locus. C . Averaged, normalized reads per million (y-axis) from mTSS-seq plotted as fragments (gray) and midpoints (black), centered on and surrounding 2kb of the TSS for ~22,000 open reading frames in hg19 (x-axis). DNase I-hypersensitivity (GSM736580; green) and RNA polymerase II from ChIP-seq (GSM935299; blue) data from A549 cells are shown. (D) LAC patient 4137 Normal nucleosomal midpoints (blue track) were plotted in the UCSC genome browser against the published human lymphocyte nucleosome distribution maps by Gaffney et. al. (green track) for the ZNF451 and CCDC97 loci. Sequence capture oligos and corresponding RefSeq gene models are shown for each locus. Correlations are shown for ZNF451 and CCDC87, respectively.

    Article Snippet: Universal and indexed sequences were added through 8 cycles of PCR, using NEBNext® Multiplex Oligos for Illumina® (Index Primers Set 1, NEB #E7335S/L).

    Techniques: Sequencing, Genome Wide, Flow Cytometry, Titration, Isolation, Chromatin Immunoprecipitation

    Visualization of infection dynamics in the JW18 cell line. (A) Schematic of Wolbachia detection by RNA Fluorescent In Situ Hybridization (FISH) using a sensitive and specific set of 48 5’-fluorescently-labeled oligos that bind in series to the 23s rRNA of the Wolbachia within a host cell. ( B) Wolbachia -infected JW18 cells labeled by 23s rRNA FISH probe can detect different infection levels in a highly specific manner. Scale bar 5μm. ( C) Wolbachia infection within the JW18 population is steadily maintained at 14% of the total cells in the population. Of the Wolbachia infected cells, the majority (73%) of cells have a low Wolbachia infection (1–10 bacteria per cell), 13.5% contain a medium Wolbachia infection (11–30 bacteria), and 13.5% of the infected JW18 cells have a high infection level ( > 30 bacteria) (n = 793 cells). See S1 – S3 Figs for further characterization of JW18 cells.

    Journal: PLoS Pathogens

    Article Title: Whole genome screen reveals a novel relationship between Wolbachia levels and Drosophila host translation

    doi: 10.1371/journal.ppat.1007445

    Figure Lengend Snippet: Visualization of infection dynamics in the JW18 cell line. (A) Schematic of Wolbachia detection by RNA Fluorescent In Situ Hybridization (FISH) using a sensitive and specific set of 48 5’-fluorescently-labeled oligos that bind in series to the 23s rRNA of the Wolbachia within a host cell. ( B) Wolbachia -infected JW18 cells labeled by 23s rRNA FISH probe can detect different infection levels in a highly specific manner. Scale bar 5μm. ( C) Wolbachia infection within the JW18 population is steadily maintained at 14% of the total cells in the population. Of the Wolbachia infected cells, the majority (73%) of cells have a low Wolbachia infection (1–10 bacteria per cell), 13.5% contain a medium Wolbachia infection (11–30 bacteria), and 13.5% of the infected JW18 cells have a high infection level ( > 30 bacteria) (n = 793 cells). See S1 – S3 Figs for further characterization of JW18 cells.

    Article Snippet: After rRNA depletion libraries were prepared according to manufacturer’s instructions using the NEBNext Ultra Directional RNA Library Prep Kit for Illumina (New England BioLabs, E7420L) and NEBNext Multiplex Oligos for Illumina Index Primers Set I (Illumina, E7335).

    Techniques: Infection, In Situ Hybridization, Fluorescence In Situ Hybridization, Labeling

    Overview of the different methods used for adapter addition to antibody variable heavy chain amplicon libraries. All methods required the reverse transcription of antibody mRNA into cDNA (step 1), which served as template for the following IgG gene-specific amplification by PCR. (A) The ligation method required a pre-amplified library as starting material, with a 3′ A-overhang added by the Taq DNA Polymerase (step 2). The stem-loop adapters containing a 5′ T-overhang were then attached in an enzymatic ligation reaction and cleaved in order to create a double-stranded form (step 3) that served as template for a final amplification step (step 4) in which the full-length Illumina TruSeq universal and index adapter sequences were incorporated into the library. (B) The direct addition method combined antibody library amplification and sequencing adapter addition into one PCR step (step 2) by attaching the Illumina adapter sequences 5′ of the gene-specific primers used for library preparation. (C) The primer extension method incorporated a GC-rich overhang into the library in PCR1 (step 2). This resulted in uniformly high amplification in a second PCR by using primers specific for the GC-rich overhang and containing the full-length Illumina sequencing adapters (step 3). UTR: untranslated region, L: leader sequence, V: variable region, C: constant region, RT: reverse transcription, fw: forward, rv: reverse, x: barcode/index allowing multiplexed sequencing runs.

    Journal: PLoS ONE

    Article Title: Comprehensive Evaluation and Optimization of Amplicon Library Preparation Methods for High-Throughput Antibody Sequencing

    doi: 10.1371/journal.pone.0096727

    Figure Lengend Snippet: Overview of the different methods used for adapter addition to antibody variable heavy chain amplicon libraries. All methods required the reverse transcription of antibody mRNA into cDNA (step 1), which served as template for the following IgG gene-specific amplification by PCR. (A) The ligation method required a pre-amplified library as starting material, with a 3′ A-overhang added by the Taq DNA Polymerase (step 2). The stem-loop adapters containing a 5′ T-overhang were then attached in an enzymatic ligation reaction and cleaved in order to create a double-stranded form (step 3) that served as template for a final amplification step (step 4) in which the full-length Illumina TruSeq universal and index adapter sequences were incorporated into the library. (B) The direct addition method combined antibody library amplification and sequencing adapter addition into one PCR step (step 2) by attaching the Illumina adapter sequences 5′ of the gene-specific primers used for library preparation. (C) The primer extension method incorporated a GC-rich overhang into the library in PCR1 (step 2). This resulted in uniformly high amplification in a second PCR by using primers specific for the GC-rich overhang and containing the full-length Illumina sequencing adapters (step 3). UTR: untranslated region, L: leader sequence, V: variable region, C: constant region, RT: reverse transcription, fw: forward, rv: reverse, x: barcode/index allowing multiplexed sequencing runs.

    Article Snippet: Parallel reactions were run to obtain ≈1 µg of gel-purified DNA library, which is the recommended minimum input for the adapter ligation kit used (NEBNext Multiplex Oligos for Illumina Kit, New England Biolabs, NEB).

    Techniques: Amplification, Polymerase Chain Reaction, Ligation, Sequencing, Gas Chromatography

    A visual representation of the circle-sequencing assay. The circle-sequencing protocol identifies transcription errors (orange circles) by fragmenting RNA (green strands) into short oligonucleotides, circularizing them, and reverse-transcribing the RNA circles in a rolling-circle reaction to generate linear cDNA molecules made up of tandem repeats of the original RNA fragment (blue strands). During this step, artificial mutations may arise in the cDNA (purple circles). The cDNA is then processed to generate a library, amplified, and sequenced, during which further artifacts may arise (teal circles). However, because these artifacts are only present in one copy of the tandem repeats, they can be distinguished from true transcription errors, which are present in all tandem repeats. bp, base pair.

    Journal: Science Advances

    Article Title: The landscape of transcription errors in eukaryotic cells

    doi: 10.1126/sciadv.1701484

    Figure Lengend Snippet: A visual representation of the circle-sequencing assay. The circle-sequencing protocol identifies transcription errors (orange circles) by fragmenting RNA (green strands) into short oligonucleotides, circularizing them, and reverse-transcribing the RNA circles in a rolling-circle reaction to generate linear cDNA molecules made up of tandem repeats of the original RNA fragment (blue strands). During this step, artificial mutations may arise in the cDNA (purple circles). The cDNA is then processed to generate a library, amplified, and sequenced, during which further artifacts may arise (teal circles). However, because these artifacts are only present in one copy of the tandem repeats, they can be distinguished from true transcription errors, which are present in all tandem repeats. bp, base pair.

    Article Snippet: These RNA molecules were then reverse-transcribed in a rolling-circle reaction according to the protocol described by Acevedo et al . , with the exception that the incubation time at 42°C was extended from 2 to 20 min. Second-strand synthesis and the remaining steps for library preparation were then performed with the NEBNext Ultra RNA Library Prep kit for Illumina (E7530L, NEB) and the NEBNext Multiplex Oligos for Illumina (E7335S and E7500S, NEB) according to the manufacturer’s protocols.

    Techniques: Sequencing, Amplification

    Comparison of ORFeome capture using LASSO or MIP probe libraries (a) Schematic of workflow of ORFeome capture using LASSO or MIP probe libraries. A genomic database was used to guide the design of the probe library, which was synthesized as pre-LASSO or pre-MIP DNA oligonucleotides on a programmable array. The pre-probe pools were converted into the mature probe pools in pooled format. The libraries of probes were hybridized on target DNA. Closed DNA circles containing captured ORFs were selected by exonuclease digestion, and then PCR amplified using universal primers. (b) Median RPKM enrichment ratios of targeted ORFs versus non-targeted genetic elements for LASSO-242bp, LASSO-442bp and MIP captures. When comparing the enrichment ratios of LASSO probes to those of MIP probes, 100 bases on either end of the ORFs were omitted for computational purposes as described further in Methods. (c) Quantification of unique ORFs cloned and sequenced from MIP and LASSO-242bp capture transformations. (d) Positions of captured reads mapped across the length-normalized target ORFs for LASSO-242bp and MIP captures. All ORFs having size > than 1kb were included in the graphs.

    Journal: Nature biomedical engineering

    Article Title: Long-adapter single-strand oligonucleotide probes for the massively multiplexed cloning of kilobase genome regions

    doi: 10.1038/s41551-017-0092

    Figure Lengend Snippet: Comparison of ORFeome capture using LASSO or MIP probe libraries (a) Schematic of workflow of ORFeome capture using LASSO or MIP probe libraries. A genomic database was used to guide the design of the probe library, which was synthesized as pre-LASSO or pre-MIP DNA oligonucleotides on a programmable array. The pre-probe pools were converted into the mature probe pools in pooled format. The libraries of probes were hybridized on target DNA. Closed DNA circles containing captured ORFs were selected by exonuclease digestion, and then PCR amplified using universal primers. (b) Median RPKM enrichment ratios of targeted ORFs versus non-targeted genetic elements for LASSO-242bp, LASSO-442bp and MIP captures. When comparing the enrichment ratios of LASSO probes to those of MIP probes, 100 bases on either end of the ORFs were omitted for computational purposes as described further in Methods. (c) Quantification of unique ORFs cloned and sequenced from MIP and LASSO-242bp capture transformations. (d) Positions of captured reads mapped across the length-normalized target ORFs for LASSO-242bp and MIP captures. All ORFs having size > than 1kb were included in the graphs.

    Article Snippet: PCR enrichment of adaptor ligated DNA was performed using NEBNext Multiplex Oligos (NEB) with index primers.

    Techniques: Synthesized, Polymerase Chain Reaction, Amplification, Clone Assay

    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.

    Journal: Oncotarget

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

    doi: 10.18632/oncotarget.6841

    Figure Lengend 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.

    Article Snippet: The universal and indexed sequences were added by PCR using 23 ul of adaptor-ligated DNA fragments, NEBNext High Fidelity 2X PCR Master Mix, index primers provided in NEBNext Multiplex (NEB #E7335, #E7500) Oligos for Illumina, and Universal PCR Primers provided in NEBNext Multiplex (NEB #E7335, #E7500) Oligos for Illumina.

    Techniques: Real-time Polymerase Chain Reaction, Sequencing