multiplex oligos  (New England Biolabs)


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  • 99
    Name:
    Oligo d T 18 no 5 Phosphate
    Description:
    Oligo d T 18 no 5 Phosphate 5 0 A260 units
    Catalog Number:
    s1316s
    Price:
    93
    Size:
    5 0 A260 units
    Category:
    Probes and Primers
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    Structured Review

    New England Biolabs multiplex oligos
    Oligo d T 18 no 5 Phosphate
    Oligo d T 18 no 5 Phosphate 5 0 A260 units
    https://www.bioz.com/result/multiplex oligos/product/New England Biolabs
    Average 99 stars, based on 14 article reviews
    Price from $9.99 to $1999.99
    multiplex oligos - by Bioz Stars, 2020-07
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    Images

    1) Product Images from "The Role of N-α-acetyltransferase 10 Protein in DNA Methylation and Genomic Imprinting"

    Article Title: The Role of N-α-acetyltransferase 10 Protein in DNA Methylation and Genomic Imprinting

    Journal: Molecular cell

    doi: 10.1016/j.molcel.2017.08.025

    Naa10p Loss Impairs Dnmt1 Activity and Binding to ICRs/DMRs (A and B) Naa10p KO reduces Dnmt1 activity in nuclear extracts of mouse ESCs. Nuclear extracts (A) or Dnmt1 immunoprecipitated (B) from two WT, two Naa10 -KO, or Dnmt1 -KO ESCs were incubated with hemi-methylated double-stranded DNA oligonucleotides and [ 3 H]-S-adenosyl-L-methionine, followed by determination of radioactivity of the DNA oligonucleotides and western blotting. After subtracting the background signal of Dnmt1 -KO cells, the relative Dnmt1 activity from each sample was normalized to the mean activity of WT replicates (A, bottom, and B, right). Data represent the mean ± SD from three independent experiments. Asterisks indicate statistical difference calculated by two-tailed t test: **p
    Figure Legend Snippet: Naa10p Loss Impairs Dnmt1 Activity and Binding to ICRs/DMRs (A and B) Naa10p KO reduces Dnmt1 activity in nuclear extracts of mouse ESCs. Nuclear extracts (A) or Dnmt1 immunoprecipitated (B) from two WT, two Naa10 -KO, or Dnmt1 -KO ESCs were incubated with hemi-methylated double-stranded DNA oligonucleotides and [ 3 H]-S-adenosyl-L-methionine, followed by determination of radioactivity of the DNA oligonucleotides and western blotting. After subtracting the background signal of Dnmt1 -KO cells, the relative Dnmt1 activity from each sample was normalized to the mean activity of WT replicates (A, bottom, and B, right). Data represent the mean ± SD from three independent experiments. Asterisks indicate statistical difference calculated by two-tailed t test: **p

    Techniques Used: Activity Assay, Binding Assay, Immunoprecipitation, Incubation, Methylation, Radioactivity, Western Blot, Two Tailed Test

    Related Articles

    Helicase Assay:

    Article Title: Action of CMG with strand-specific DNA blocks supports an internal unwinding mode for the eukaryotic replicative helicase
    Article Snippet: .. Helicase assay substrates For all radiolabeled oligos, 10 pmols of oligo were labeled at the 5’ terminus with 0.05 mCi [γ-32 P]-ATP using T4 Polynucleotide Kinase (New England Biolabs) in a 25 μl reaction for 30’ @ 37°C according to the manufacturer’s instructions. .. For annealing, 4 pmols of the radiolabeled strand were mixed with 6 pmols of the unlabeled complementary strand, NaCl was added to a final concentration of 200 mM, and the mixture was heated to 90°C and cooled slowly to room temperature.

    Labeling:

    Article Title: Action of CMG with strand-specific DNA blocks supports an internal unwinding mode for the eukaryotic replicative helicase
    Article Snippet: .. Helicase assay substrates For all radiolabeled oligos, 10 pmols of oligo were labeled at the 5’ terminus with 0.05 mCi [γ-32 P]-ATP using T4 Polynucleotide Kinase (New England Biolabs) in a 25 μl reaction for 30’ @ 37°C according to the manufacturer’s instructions. .. For annealing, 4 pmols of the radiolabeled strand were mixed with 6 pmols of the unlabeled complementary strand, NaCl was added to a final concentration of 200 mM, and the mixture was heated to 90°C and cooled slowly to room temperature.

    Article Title: Molecular Factors Affecting the Accumulation of Recombinant Proteins in the Chlamydomonas reinhardtii Chloroplast
    Article Snippet: .. Twenty picomoles of the following oligos: 5′-gcaataccgtcacctacttgg-3′ (for atpA ) and 5′-ccataagttgcgtcacc-3′ (for gfp ) were labeled with T4 polynucleotide kinase (20 U, New England Biolabs, Ipswich, MA) according to the manufacturer’s instructions. .. Labeled oligos were purified from unincorporated radioactive isotope using Quick Spin Sephadex G-25 columns (Roche).

    Magnetic Beads:

    Article Title: An mRNA-binding channel in the ES6S region of the translation 48S-PIC promotes RNA unwinding and scanning
    Article Snippet: .. After 30 min crosslinking at 254 nm, WRFs were resuspended in buffer D (Tris-HCl 30 mM [pH 7.5], 0.5 M LiCl, 0.5% LiDS, 0.5 mM EDTA and 1 mM DTT) and poly(A)+ mRNA was captured with oligo(dT) magnetic beads (NEB) under denaturing conditions as described above. ..

    Article Title: Local regulation of gene expression by lncRNA promoters, transcription, and splicing
    Article Snippet: .. We enriched for poly(A)+ RNA using oligo d(T)25 magnetic beads (NEB) and eluted in 18 μl H2 O. .. We fragmented RNA to an average of ~150-nt by adding 2 μl Ambion Fragmentation Buffer and incubating at 70°C for exactly 2.5 minutes.

    Article Title: An mRNA-binding channel in the ES6S region of the translation 48S-PIC promotes RNA unwinding and scanning
    Article Snippet: .. After UV crosslinking at 360 nm, the WRF was denatured in 500 μl buffer D (Tris-HCl 30 mM [pH 7.5], 0.5 M LiCl, 0.5% LiDS, 0.5 mM EDTA and 1 mM DTT) and poly(A)+ mRNA was captured with oligo(dT) magnetic beads (NEB) under denaturing conditions. .. After extensive washing, RNA was eluted with 100 μl H2 O and concentrated by ethanol precipitation in the presence of glycogen.

    Article Title: An RNA trapping mechanism in Alphavirus mRNA promotes ribosome stalling and translation initiation
    Article Snippet: .. The complexes were crosslinked, centrifuged at high speed and ribosomal pellets were resuspended in cracking buffer (25 mM Tris-HCl pH 7.5, 0.5 M LiCl, 0.5% LiDS, 1 mM EDTA and 5 mM DTT). mRNA poly(A)+ was bound to oligo (dT)25 magnetic beads (NEB) at RT for 20 min. Beads were extensively washed according to the manufacturer's recommendations and eluted in TE buffer by heating the samples at 60°C for 10 min. RNA was extracted with phenol, precipitated with ethanol and resuspended in 15 μl of TE buffer. ..

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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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    85
    New England Biolabs sense fn14 primer targeting exon 1 nucleotides 150
    Cloning of a human <t>Fn14</t> mRNA predicted to encode an Fn14 protein missing most of the extracellular domain. ( A ) Schematic representation of human Fn14 gene organization (via UCSC Genome Browser). The four Fn14 exons are numbered and boxed and the intron sizes are indicated in nucleotides (nt) at the top. The Fn14 amino acid (aa) numbers (1–129) and mature mRNA nucleotide (nt) numbers (1–1033) are provided above or below each exon, respectively. The positions of the two oligonucleotide primers used for RT-PCR analysis are indicated with arrows. ( B ) RNA was isolated from MDA-MB-231 and U87 cells and RT-PCR was performed using an <t>exon</t> 1 sense primer and an exon 4 antisense primer. PCR was also performed with this primer pair in the absence of cDNA (NT, no template). Amplification products were separated by agarose gel electrophoresis and visualized by ethidium bromide staining. The positions of DNA size markers (M) are shown on the left (in base pairs). The two PCR products that were isolated and sequenced are indicated on the right as a and b. ( C ) Predicted amino acid sequence of PCR amplification products a and b. The last six amino acids of the signal peptide are indicated with an arrow, the Fn14 extracellular domain is bracketed with asterisks and the six cysteine residues found in this domain are in red. The Fn14 transmembrane domain is overlined and the cytoplasmic tail is underlined. ( D ) The Fn14 mRNA translation initiation codon and selected codons surrounding Fn14 introns 1 and 2 are shown for the Fn14 full-length (Fn14-FL) and Fn14 extracellular domain deletion (Fn14-ΔEC) mRNAs. The predicted Fn14-FL and Fn14-ΔEC amino acid sequence is shown below the nucleotide sequence. Abbreviation: I, intron. ( E ) Schematic representation of the Fn14-FL and Fn14-ΔEC proteins showing structural domains in relation to their exon coding regions. Amino acid numbers corresponding to the beginning of each protein domain and the C-terminal amino acid are indicated at the top of each diagram. The region of the extracellular domain that is missing in Fn14-ΔEC is shown in black. Abbreviations: SP, signal peptide; EC, extracellular domain; TM, transmembrane domain; CYT, cytoplasmic tail.
    Sense Fn14 Primer Targeting Exon 1 Nucleotides 150, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 85/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    New England Biolabs spei
    Over-expressing L1-ectodomain in MDA-MB-468 cells . (A) Schematic diagram of Lvv 1879 vector containing L1ED. 3350 bp L1 ectodomain fragment was amplified and inserted into Lvv 1879 via <t>SpeI</t> and <t>XhoI</t> restriction enzyme sites. The constructed lentivirus was used to infect MDA-MB-468 cells to establish a new stable cell line. (B) Immunostaining and FACS analysis of L1CAM level in MDA-MB-468-L1ED compared to mock vector infected and plain MDA-MB-468 cells. (C) TCA precipitation and western blotting examining over-expressed L1 ectodomain release in MDA-MB-468-L1ED culture medium by monoclonal antibody 5G3. The amount of cell associated L1 in pellets was probed by polyclonal antibody NCAM-L1 (C-20).
    Spei, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 99/100, based on 249 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Image Search Results


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

    Journal: Nucleic Acids Research

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

    doi: 10.1093/nar/gky1305

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

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

    Techniques: Isolation, Selection

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

    Journal: Nucleic Acids Research

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

    doi: 10.1093/nar/gky1305

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

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

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

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

    Journal: Nucleic Acids Research

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

    doi: 10.1093/nar/gky1305

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

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

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

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

    Journal: Nucleic Acids Research

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

    doi: 10.1093/nar/gky1305

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

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

    Techniques: Isolation

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

    Journal: Nucleic Acids Research

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

    doi: 10.1093/nar/gky1305

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

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

    Techniques: Whisker Assay, Labeling

    Cloning of a human Fn14 mRNA predicted to encode an Fn14 protein missing most of the extracellular domain. ( A ) Schematic representation of human Fn14 gene organization (via UCSC Genome Browser). The four Fn14 exons are numbered and boxed and the intron sizes are indicated in nucleotides (nt) at the top. The Fn14 amino acid (aa) numbers (1–129) and mature mRNA nucleotide (nt) numbers (1–1033) are provided above or below each exon, respectively. The positions of the two oligonucleotide primers used for RT-PCR analysis are indicated with arrows. ( B ) RNA was isolated from MDA-MB-231 and U87 cells and RT-PCR was performed using an exon 1 sense primer and an exon 4 antisense primer. PCR was also performed with this primer pair in the absence of cDNA (NT, no template). Amplification products were separated by agarose gel electrophoresis and visualized by ethidium bromide staining. The positions of DNA size markers (M) are shown on the left (in base pairs). The two PCR products that were isolated and sequenced are indicated on the right as a and b. ( C ) Predicted amino acid sequence of PCR amplification products a and b. The last six amino acids of the signal peptide are indicated with an arrow, the Fn14 extracellular domain is bracketed with asterisks and the six cysteine residues found in this domain are in red. The Fn14 transmembrane domain is overlined and the cytoplasmic tail is underlined. ( D ) The Fn14 mRNA translation initiation codon and selected codons surrounding Fn14 introns 1 and 2 are shown for the Fn14 full-length (Fn14-FL) and Fn14 extracellular domain deletion (Fn14-ΔEC) mRNAs. The predicted Fn14-FL and Fn14-ΔEC amino acid sequence is shown below the nucleotide sequence. Abbreviation: I, intron. ( E ) Schematic representation of the Fn14-FL and Fn14-ΔEC proteins showing structural domains in relation to their exon coding regions. Amino acid numbers corresponding to the beginning of each protein domain and the C-terminal amino acid are indicated at the top of each diagram. The region of the extracellular domain that is missing in Fn14-ΔEC is shown in black. Abbreviations: SP, signal peptide; EC, extracellular domain; TM, transmembrane domain; CYT, cytoplasmic tail.

    Journal: PLoS ONE

    Article Title: TWEAK-Independent Fn14 Self-Association and NF-?B Activation Is Mediated by the C-Terminal Region of the Fn14 Cytoplasmic Domain

    doi: 10.1371/journal.pone.0065248

    Figure Lengend Snippet: Cloning of a human Fn14 mRNA predicted to encode an Fn14 protein missing most of the extracellular domain. ( A ) Schematic representation of human Fn14 gene organization (via UCSC Genome Browser). The four Fn14 exons are numbered and boxed and the intron sizes are indicated in nucleotides (nt) at the top. The Fn14 amino acid (aa) numbers (1–129) and mature mRNA nucleotide (nt) numbers (1–1033) are provided above or below each exon, respectively. The positions of the two oligonucleotide primers used for RT-PCR analysis are indicated with arrows. ( B ) RNA was isolated from MDA-MB-231 and U87 cells and RT-PCR was performed using an exon 1 sense primer and an exon 4 antisense primer. PCR was also performed with this primer pair in the absence of cDNA (NT, no template). Amplification products were separated by agarose gel electrophoresis and visualized by ethidium bromide staining. The positions of DNA size markers (M) are shown on the left (in base pairs). The two PCR products that were isolated and sequenced are indicated on the right as a and b. ( C ) Predicted amino acid sequence of PCR amplification products a and b. The last six amino acids of the signal peptide are indicated with an arrow, the Fn14 extracellular domain is bracketed with asterisks and the six cysteine residues found in this domain are in red. The Fn14 transmembrane domain is overlined and the cytoplasmic tail is underlined. ( D ) The Fn14 mRNA translation initiation codon and selected codons surrounding Fn14 introns 1 and 2 are shown for the Fn14 full-length (Fn14-FL) and Fn14 extracellular domain deletion (Fn14-ΔEC) mRNAs. The predicted Fn14-FL and Fn14-ΔEC amino acid sequence is shown below the nucleotide sequence. Abbreviation: I, intron. ( E ) Schematic representation of the Fn14-FL and Fn14-ΔEC proteins showing structural domains in relation to their exon coding regions. Amino acid numbers corresponding to the beginning of each protein domain and the C-terminal amino acid are indicated at the top of each diagram. The region of the extracellular domain that is missing in Fn14-ΔEC is shown in black. Abbreviations: SP, signal peptide; EC, extracellular domain; TM, transmembrane domain; CYT, cytoplasmic tail.

    Article Snippet: Second, PCR was performed in the presence of 5% DMSO using a sense Fn14 primer targeting exon 1 nucleotides 150–176, an antisense primer targeting exon 4 nucleotides 30–56, and Vent Polymerase (New England Biolabs).

    Techniques: Clone Assay, Reverse Transcription Polymerase Chain Reaction, Isolation, Multiple Displacement Amplification, Polymerase Chain Reaction, Amplification, Agarose Gel Electrophoresis, Staining, Sequencing

    Over-expressing L1-ectodomain in MDA-MB-468 cells . (A) Schematic diagram of Lvv 1879 vector containing L1ED. 3350 bp L1 ectodomain fragment was amplified and inserted into Lvv 1879 via SpeI and XhoI restriction enzyme sites. The constructed lentivirus was used to infect MDA-MB-468 cells to establish a new stable cell line. (B) Immunostaining and FACS analysis of L1CAM level in MDA-MB-468-L1ED compared to mock vector infected and plain MDA-MB-468 cells. (C) TCA precipitation and western blotting examining over-expressed L1 ectodomain release in MDA-MB-468-L1ED culture medium by monoclonal antibody 5G3. The amount of cell associated L1 in pellets was probed by polyclonal antibody NCAM-L1 (C-20).

    Journal: Cancer Cell International

    Article Title: Soluble L1CAM promotes breast cancer cell adhesion and migration in vitro, but not invasion

    doi: 10.1186/1475-2867-10-34

    Figure Lengend Snippet: Over-expressing L1-ectodomain in MDA-MB-468 cells . (A) Schematic diagram of Lvv 1879 vector containing L1ED. 3350 bp L1 ectodomain fragment was amplified and inserted into Lvv 1879 via SpeI and XhoI restriction enzyme sites. The constructed lentivirus was used to infect MDA-MB-468 cells to establish a new stable cell line. (B) Immunostaining and FACS analysis of L1CAM level in MDA-MB-468-L1ED compared to mock vector infected and plain MDA-MB-468 cells. (C) TCA precipitation and western blotting examining over-expressed L1 ectodomain release in MDA-MB-468-L1ED culture medium by monoclonal antibody 5G3. The amount of cell associated L1 in pellets was probed by polyclonal antibody NCAM-L1 (C-20).

    Article Snippet: The following primers with SpeI or XhoI (NEB, Ipswich, MA) cleavage sites were used to amplify the sequence: sense, 5'-GAAACTAGTCGCCGGGAAAG-3'; antisense, 5'- GCCTCGAGGAGGGAGCC- 3'.

    Techniques: Expressing, Multiple Displacement Amplification, Plasmid Preparation, Amplification, Construct, Stable Transfection, Immunostaining, FACS, Infection, TCA Precipitation, Western Blot