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    NEBNext Ultra II DNA Library Prep Kit for Illumina
    Description:
    NEBNext Ultra II DNA Library Prep Kit for Illumina 96 rxns
    Catalog Number:
    e7645l
    Price:
    2245
    Size:
    96 rxns
    Category:
    DNA Template Preparation for PCR
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    New England Biolabs nebnext multiplex oligos
    NEBNext Ultra II DNA Library Prep Kit for Illumina
    NEBNext Ultra II DNA Library Prep Kit for Illumina 96 rxns
    https://www.bioz.com/result/nebnext multiplex oligos/product/New England Biolabs
    Average 99 stars, based on 219 article reviews
    Price from $9.99 to $1999.99
    nebnext multiplex oligos - by Bioz Stars, 2020-07
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    Multiplex Assay:

    Article Title: Isolation and genome-wide characterization of cellular DNA:RNA triplex structures
    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). .. Single-end sequencing was performed on Illumina NextSeq 500 platform.

    Ligation:

    Article Title: RELACS nuclei barcoding enables high-throughput ChIP-seq
    Article Snippet: .. Ligation of barcodes to A-tailed chromatin was performed using components from NEBNext Ultra II DNA library preparation kit, with the following modifications: per sample 15 µL of ligation master mix and 0.5 µL of ligation enhancer were added, followed by 15 min incubation at 30 °C and 15 min incubation at 20 °C. ..

    Article Title: NickSeq for genome-wide strand-specific identification of DNA single-strand break sites with single nucleotide resolution
    Article Snippet: .. Human genomic DNA was fragmented by a dsDNA fragmentase (New England Biolabs) followed by end preparation and adapter ligation according to the NEBNext Ultra II DNA Library Prep Method for Illumina (New England Biolabs). .. 25 μg of Dynabeads MyOne Streptavidin C1 (Invitrogen) were washed in 100 μL of bind and wash buffer, 1 M sodium chloride and 0.1% tween-20 in TE buffer.

    Purification:

    Article Title: Avoidance of Trinucleotide Corresponding to Consensus Protospacer Adjacent Motif Controls the Efficiency of Prespacer Selection during Primed Adaptation
    Article Snippet: .. Amplicons containing plasmid prespacers and extended CRISPR arrays were gel purified and used to create Illumina sequencing libraries with an NEBNext Ultra II DNA library preparation kit with U5 barcoding. .. High-throughput sequencing of amplicons was conducted on MiniSeq or HiSeq Illumina machines using the 2 × 150 paired-end mode.

    Real-time Polymerase Chain Reaction:

    Article Title: Sister-chromatid-sensitive Hi-C reveals the conformation of replicated human chromosomes
    Article Snippet: .. Then, 4sT was converted to methyl-cytosine using OsO4 / NH4 Cl (see below), followed by qPCR according the NEBUltra Ultra II DNA library prep kit for Illumina [NEB]. .. The finished libraries were purified using AMPure XP beads (Beckman Coulter) at 0.9x sample volume following the standard protocol.

    Incubation:

    Article Title: Solid-phase enzyme catalysis of DNA end repair and 3′ A-tailing reduces GC-bias in next-generation sequencing of human genomic DNA
    Article Snippet: .. Similar to the method using PKT, the protocols of NEBNext Ultra II DNA Library Prep Kit and Kapa Hyper Prep Kit adopt high temperature incubation at 65 °C (Fig. ). .. The protocol of Illumina TruSeq DNA PCR-free LT Library Preparation Kit includes a 5 min incubation at 70 °C following 3′ A-tailing for 30 min at 37 °C (Fig. ).

    Article Title: RELACS nuclei barcoding enables high-throughput ChIP-seq
    Article Snippet: .. Ligation of barcodes to A-tailed chromatin was performed using components from NEBNext Ultra II DNA library preparation kit, with the following modifications: per sample 15 µL of ligation master mix and 0.5 µL of ligation enhancer were added, followed by 15 min incubation at 30 °C and 15 min incubation at 20 °C. ..

    CRISPR:

    Article Title: Avoidance of Trinucleotide Corresponding to Consensus Protospacer Adjacent Motif Controls the Efficiency of Prespacer Selection during Primed Adaptation
    Article Snippet: .. Amplicons containing plasmid prespacers and extended CRISPR arrays were gel purified and used to create Illumina sequencing libraries with an NEBNext Ultra II DNA library preparation kit with U5 barcoding. .. High-throughput sequencing of amplicons was conducted on MiniSeq or HiSeq Illumina machines using the 2 × 150 paired-end mode.

    Sequencing:

    Article Title: Avoidance of Trinucleotide Corresponding to Consensus Protospacer Adjacent Motif Controls the Efficiency of Prespacer Selection during Primed Adaptation
    Article Snippet: .. Amplicons containing plasmid prespacers and extended CRISPR arrays were gel purified and used to create Illumina sequencing libraries with an NEBNext Ultra II DNA library preparation kit with U5 barcoding. .. High-throughput sequencing of amplicons was conducted on MiniSeq or HiSeq Illumina machines using the 2 × 150 paired-end mode.

    Plasmid Preparation:

    Article Title: Avoidance of Trinucleotide Corresponding to Consensus Protospacer Adjacent Motif Controls the Efficiency of Prespacer Selection during Primed Adaptation
    Article Snippet: .. Amplicons containing plasmid prespacers and extended CRISPR arrays were gel purified and used to create Illumina sequencing libraries with an NEBNext Ultra II DNA library preparation kit with U5 barcoding. .. High-throughput sequencing of amplicons was conducted on MiniSeq or HiSeq Illumina machines using the 2 × 150 paired-end mode.

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    New England Biolabs ultra ii dna library prep kit
    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
    Ultra Ii Dna Library Prep Kit, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 99/100, based on 68 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    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 16 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/universal pcr primers/product/New England Biolabs
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    New England Biolabs nebnext multiplex oligos for illumina
    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 <t>Illumina</t> sequencing. Solution-based sequence capture is performed using biotinylated <t>oligos,</t> 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.
    Nebnext Multiplex Oligos For Illumina, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 99/100, based on 309 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    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

    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

    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, Titration, Isolation, Chromatin Immunoprecipitation