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  • 99
    New England Biolabs multiplex oligos
    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 Oligos, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 99/100, based on 29 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    83
    Pacific Biosciences multiplex pcr primer guidelines
    Mutations affecting PgABCA2 protein in Cry2Ab-resistant pink bollworm from Arizona and India. ( a ) The predicted PgABCA2 protein includes amino (N) and carboxyl (C) termini (pink), two transmembrane domains (TMD1 and TMD2), each consisting of 6 transmembrane regions (TM; orange), three extracellular loops (ECL; green), two intracellular loops (ICL; blue), and two nucleotide-binding domains (NBD; purple). Mutations affecting transcripts of resistant pink bollworm: Circles show premature stop codons from India (red), Arizona (yellow), or both (red and yellow). Triangles show in-frame indels from India (red) or Arizona (yellow). Numbers indicate the affected amino acids. ( b ) Full-length and partial PgABCA2 cDNAs were obtained by direct <t>PCR</t> sequencing, DNA sequencing of cDNA clones, and/or <t>PacBio</t> ® DNA sequencing from susceptible (APHIS-S) and resistant, laboratory-selected (Bt4-R2) pink bollworm from Arizona, USA and India field-selected resistant populations (AM, CK, GAP, KT, and RK). The linear schematic (top) shows the predicted translated domain structure of the 5,187-bp full-length PgABCA2 coding sequence. The predicted protein includes amino- and carboxyl-termini (pink), transmembrane regions TM1-TM12 (orange), intracellular loops ICL1-ICL5 (blue), and extracellular loops ECL1-ECL6 (green). The domain structure connected to the exons that encode the respective domains is shown by dotted gray lines. Each predicted domain is numbered, with ECLs on top and ICLs numbered on bottom of protein schematic. Putative exons 1–31 are numbered, with grey exons indicating regions determined by direct PCR sequencing. Exons colored in light blue were further verified by sequencing cDNA clones (either by Sanger or PacBio ® sequencing). Red bars indicate disruption sites within the full-length coding sequence and the red triangles indicate the location of premature stop codons shown to scale based on the linear schematic of the translated domain structure. Unique cDNA variants are indicated as a, b, c, etc.
    Multiplex Pcr Primer Guidelines, supplied by Pacific Biosciences, used in various techniques. Bioz Stars score: 83/100, based on 12 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    79
    Sequenom multiplex rna single nucleotide primer extension snupe assays
    Testing for transgenerational epigenetic inheritance of the aberrant imprinted expression. (A) Breeding design to test whether ED-perturbed parental allele-specific transcription is transgenerationally inherited through the paternal germline to an unexposed generation. G1 male fetuses were exposed in utero to EDs or vehicle control (‘oil’) daily from 12.5 dpc to 16.5 dpc. After reaching adulthood, 129S1 G1 males were mated with 129S1 unexposed females to generate G2 offspring (3 blue stars), which derived from exposed prospermatogonia. At adulthood, G2 males were mated with unexposed JF1 females to generate G3 offspring, which were never directly exposed to EDs. JF1 × 129 G3 fetuses were dissected at 13.5 dpc to collect organs for <t>RNA</t> isolation. Parental-specific transcription was quantified in the total RNA using multiplex <t>SNuPE</t> assays. (B) Results of Sequenom allelotyping experiments using heart and lung tissue of the G3 generation; color scale as in Figure 2 ; letters in parentheses denote independent SNPs. Notice the lack of inherited changes from the exposed generation. More groups of fetuses are shown in Additional file 2 . This Figure includes standards that are routinely included in the Sequenom runs (see Methods).
    Multiplex Rna Single Nucleotide Primer Extension Snupe Assays, supplied by Sequenom, used in various techniques. Bioz Stars score: 79/100, based on 5 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    78
    Millipore multiple pyk2 sirna oligonucleotides
    <t>Pyk2</t> activation is required for stimulation of cell motility by ATP. (A) HCLE cells were transfected with control or Pyk2 <t>siRNA</t> prior to the wound healing assays, and were incubated with ATP as indicated. P values for relevant significant differences
    Multiple Pyk2 Sirna Oligonucleotides, supplied by Millipore, used in various techniques. Bioz Stars score: 78/100, based on 9 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    78
    Agilent technologies whole human genome 4x44 multiplex format oligonucleotide arrays
    Dex and E 2 regulate global gene expression in human uterine epithelial cells. A ) mRNA isolated from Dex-, E 2 -, and Dex + E 2 -treated ECC1 cells was analyzed using Whole Human Genome <t>4x44</t> multiplex format oligo array (Agilent) for gene expression. A heat
    Whole Human Genome 4x44 Multiplex Format Oligonucleotide Arrays, supplied by Agilent technologies, used in various techniques. Bioz Stars score: 78/100, based on 5 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    76
    Agilent technologies multiple long oligonucleotide based microarray platforms
    Top regulated genes derived from meta-analysis . RankProd analysis of the combination of <t>microarray</t> and Illumina GA-I ultrasequencing data sets. Heatmap of the top 50 up and down-regulated genes detected in all four platforms ordered by Median Fold Change (all have RankProd adjusted p-values
    Multiple Long Oligonucleotide Based Microarray Platforms, supplied by Agilent technologies, used in various techniques. Bioz Stars score: 76/100, based on 2 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

    Mutations affecting PgABCA2 protein in Cry2Ab-resistant pink bollworm from Arizona and India. ( a ) The predicted PgABCA2 protein includes amino (N) and carboxyl (C) termini (pink), two transmembrane domains (TMD1 and TMD2), each consisting of 6 transmembrane regions (TM; orange), three extracellular loops (ECL; green), two intracellular loops (ICL; blue), and two nucleotide-binding domains (NBD; purple). Mutations affecting transcripts of resistant pink bollworm: Circles show premature stop codons from India (red), Arizona (yellow), or both (red and yellow). Triangles show in-frame indels from India (red) or Arizona (yellow). Numbers indicate the affected amino acids. ( b ) Full-length and partial PgABCA2 cDNAs were obtained by direct PCR sequencing, DNA sequencing of cDNA clones, and/or PacBio ® DNA sequencing from susceptible (APHIS-S) and resistant, laboratory-selected (Bt4-R2) pink bollworm from Arizona, USA and India field-selected resistant populations (AM, CK, GAP, KT, and RK). The linear schematic (top) shows the predicted translated domain structure of the 5,187-bp full-length PgABCA2 coding sequence. The predicted protein includes amino- and carboxyl-termini (pink), transmembrane regions TM1-TM12 (orange), intracellular loops ICL1-ICL5 (blue), and extracellular loops ECL1-ECL6 (green). The domain structure connected to the exons that encode the respective domains is shown by dotted gray lines. Each predicted domain is numbered, with ECLs on top and ICLs numbered on bottom of protein schematic. Putative exons 1–31 are numbered, with grey exons indicating regions determined by direct PCR sequencing. Exons colored in light blue were further verified by sequencing cDNA clones (either by Sanger or PacBio ® sequencing). Red bars indicate disruption sites within the full-length coding sequence and the red triangles indicate the location of premature stop codons shown to scale based on the linear schematic of the translated domain structure. Unique cDNA variants are indicated as a, b, c, etc.

    Journal: Scientific Reports

    Article Title: ABC transporter mis-splicing associated with resistance to Bt toxin Cry2Ab in laboratory- and field-selected pink bollworm

    doi: 10.1038/s41598-018-31840-5

    Figure Lengend Snippet: Mutations affecting PgABCA2 protein in Cry2Ab-resistant pink bollworm from Arizona and India. ( a ) The predicted PgABCA2 protein includes amino (N) and carboxyl (C) termini (pink), two transmembrane domains (TMD1 and TMD2), each consisting of 6 transmembrane regions (TM; orange), three extracellular loops (ECL; green), two intracellular loops (ICL; blue), and two nucleotide-binding domains (NBD; purple). Mutations affecting transcripts of resistant pink bollworm: Circles show premature stop codons from India (red), Arizona (yellow), or both (red and yellow). Triangles show in-frame indels from India (red) or Arizona (yellow). Numbers indicate the affected amino acids. ( b ) Full-length and partial PgABCA2 cDNAs were obtained by direct PCR sequencing, DNA sequencing of cDNA clones, and/or PacBio ® DNA sequencing from susceptible (APHIS-S) and resistant, laboratory-selected (Bt4-R2) pink bollworm from Arizona, USA and India field-selected resistant populations (AM, CK, GAP, KT, and RK). The linear schematic (top) shows the predicted translated domain structure of the 5,187-bp full-length PgABCA2 coding sequence. The predicted protein includes amino- and carboxyl-termini (pink), transmembrane regions TM1-TM12 (orange), intracellular loops ICL1-ICL5 (blue), and extracellular loops ECL1-ECL6 (green). The domain structure connected to the exons that encode the respective domains is shown by dotted gray lines. Each predicted domain is numbered, with ECLs on top and ICLs numbered on bottom of protein schematic. Putative exons 1–31 are numbered, with grey exons indicating regions determined by direct PCR sequencing. Exons colored in light blue were further verified by sequencing cDNA clones (either by Sanger or PacBio ® sequencing). Red bars indicate disruption sites within the full-length coding sequence and the red triangles indicate the location of premature stop codons shown to scale based on the linear schematic of the translated domain structure. Unique cDNA variants are indicated as a, b, c, etc.

    Article Snippet: The symmetric barcode-tailed PCR primers were designed based on the PacBio® multiplex PCR primer guidelines ( http://www.2einteractive.com/pacbio/Shared-Protocol-PacBio-Barcodes-for-SMRT-Sequencing.pdf ).

    Techniques: Binding Assay, Polymerase Chain Reaction, Sequencing, DNA Sequencing, Clone Assay

    Testing for transgenerational epigenetic inheritance of the aberrant imprinted expression. (A) Breeding design to test whether ED-perturbed parental allele-specific transcription is transgenerationally inherited through the paternal germline to an unexposed generation. G1 male fetuses were exposed in utero to EDs or vehicle control (‘oil’) daily from 12.5 dpc to 16.5 dpc. After reaching adulthood, 129S1 G1 males were mated with 129S1 unexposed females to generate G2 offspring (3 blue stars), which derived from exposed prospermatogonia. At adulthood, G2 males were mated with unexposed JF1 females to generate G3 offspring, which were never directly exposed to EDs. JF1 × 129 G3 fetuses were dissected at 13.5 dpc to collect organs for RNA isolation. Parental-specific transcription was quantified in the total RNA using multiplex SNuPE assays. (B) Results of Sequenom allelotyping experiments using heart and lung tissue of the G3 generation; color scale as in Figure 2 ; letters in parentheses denote independent SNPs. Notice the lack of inherited changes from the exposed generation. More groups of fetuses are shown in Additional file 2 . This Figure includes standards that are routinely included in the Sequenom runs (see Methods).

    Journal: Genome Biology

    Article Title: Deleterious effects of endocrine disruptors are corrected in the mammalian germline by epigenome reprogramming

    doi: 10.1186/s13059-015-0619-z

    Figure Lengend Snippet: Testing for transgenerational epigenetic inheritance of the aberrant imprinted expression. (A) Breeding design to test whether ED-perturbed parental allele-specific transcription is transgenerationally inherited through the paternal germline to an unexposed generation. G1 male fetuses were exposed in utero to EDs or vehicle control (‘oil’) daily from 12.5 dpc to 16.5 dpc. After reaching adulthood, 129S1 G1 males were mated with 129S1 unexposed females to generate G2 offspring (3 blue stars), which derived from exposed prospermatogonia. At adulthood, G2 males were mated with unexposed JF1 females to generate G3 offspring, which were never directly exposed to EDs. JF1 × 129 G3 fetuses were dissected at 13.5 dpc to collect organs for RNA isolation. Parental-specific transcription was quantified in the total RNA using multiplex SNuPE assays. (B) Results of Sequenom allelotyping experiments using heart and lung tissue of the G3 generation; color scale as in Figure 2 ; letters in parentheses denote independent SNPs. Notice the lack of inherited changes from the exposed generation. More groups of fetuses are shown in Additional file 2 . This Figure includes standards that are routinely included in the Sequenom runs (see Methods).

    Article Snippet: This allowed measurement of the allele-specific transcription of known imprinted genes in JF1 × OG2 cells using multiplex RNA-single nucleotide primer extension (SNuPE) assays uisng Sequenom allelotyping [ ].

    Techniques: Expressing, In Utero, Derivative Assay, Isolation, Multiplex Assay

    Pyk2 activation is required for stimulation of cell motility by ATP. (A) HCLE cells were transfected with control or Pyk2 siRNA prior to the wound healing assays, and were incubated with ATP as indicated. P values for relevant significant differences

    Journal:

    Article Title: Extracellular ATP stimulates epithelial cell motility through Pyk2-mediated activation of the EGF receptor

    doi: 10.1016/j.cellsig.2011.07.021

    Figure Lengend Snippet: Pyk2 activation is required for stimulation of cell motility by ATP. (A) HCLE cells were transfected with control or Pyk2 siRNA prior to the wound healing assays, and were incubated with ATP as indicated. P values for relevant significant differences

    Article Snippet: Multiple Pyk2 siRNA oligonucleotides were used: CACAUGAAGUCCGAUGAGAdTdT (Sigma) and a Pyk2 SMART pool that contains at least four duplexes of undisclosed sequence (Millipore).

    Techniques: Activation Assay, Transfection, Incubation

    Knockdown of Pyk2 with siRNA inhibits EGFR activation after ATP stimulation but not after stimulation with EGF. (A) HCLE cells transfected with 10 nM of control or Pyk2 siRNA were treated with ATP as indicated. Immunoblots were probed for EGFR phosphorylated

    Journal:

    Article Title: Extracellular ATP stimulates epithelial cell motility through Pyk2-mediated activation of the EGF receptor

    doi: 10.1016/j.cellsig.2011.07.021

    Figure Lengend Snippet: Knockdown of Pyk2 with siRNA inhibits EGFR activation after ATP stimulation but not after stimulation with EGF. (A) HCLE cells transfected with 10 nM of control or Pyk2 siRNA were treated with ATP as indicated. Immunoblots were probed for EGFR phosphorylated

    Article Snippet: Multiple Pyk2 siRNA oligonucleotides were used: CACAUGAAGUCCGAUGAGAdTdT (Sigma) and a Pyk2 SMART pool that contains at least four duplexes of undisclosed sequence (Millipore).

    Techniques: Activation Assay, Transfection, Western Blot

    Dex and E 2 regulate global gene expression in human uterine epithelial cells. A ) mRNA isolated from Dex-, E 2 -, and Dex + E 2 -treated ECC1 cells was analyzed using Whole Human Genome 4x44 multiplex format oligo array (Agilent) for gene expression. A heat

    Journal:

    Article Title: Global Gene Expression Analysis in Human Uterine Epithelial Cells Defines New Targets of Glucocorticoid and Estradiol Antagonism

    doi: 10.1095/biolreprod.113.111054

    Figure Lengend Snippet: Dex and E 2 regulate global gene expression in human uterine epithelial cells. A ) mRNA isolated from Dex-, E 2 -, and Dex + E 2 -treated ECC1 cells was analyzed using Whole Human Genome 4x44 multiplex format oligo array (Agilent) for gene expression. A heat

    Article Snippet: Gene expression analysis was conducted using whole Human Genome 4x44 multiplex format oligonucleotide arrays (product no. 014850; Agilent Technologies) following the Agilent one-color microarray-based gene expression analysis protocol.

    Techniques: Expressing, Isolation, Multiplex Assay

    Top regulated genes derived from meta-analysis . RankProd analysis of the combination of microarray and Illumina GA-I ultrasequencing data sets. Heatmap of the top 50 up and down-regulated genes detected in all four platforms ordered by Median Fold Change (all have RankProd adjusted p-values

    Journal: BMC Genomics

    Article Title: Multiple platform assessment of the EGF dependent transcriptome by microarray and deep tag sequencing analysis

    doi: 10.1186/1471-2164-12-326

    Figure Lengend Snippet: Top regulated genes derived from meta-analysis . RankProd analysis of the combination of microarray and Illumina GA-I ultrasequencing data sets. Heatmap of the top 50 up and down-regulated genes detected in all four platforms ordered by Median Fold Change (all have RankProd adjusted p-values

    Article Snippet: Here we use a combined approach to study the EGF dependent transcriptome of HeLa cells by using multiple long oligonucleotide based microarray platforms (from Agilent, Operon, and Illumina) in combination with digital gene expression profiling (DGE) with the Illumina Genome Analyzer.

    Techniques: Derivative Assay, Microarray

    GSEA analysis on significantly regulated gene sets across microarray platforms . Profile of the Running ES Score Positions of Gene Set Members on the Rank Ordered List using 6 h EGF treatment data according to each of the three microarray platforms. In each panel, the vertical black lines indicate the position of each of the genes of the tested gene set in the reference data set (ranked by average of the three respective EGF versus control log2ratios of replicate experiments). The green curve plots the ES (enrichment score), which is the running sum of the weighted enrichment score obtained from GSEA software. Within each queried gene set, the farther the position of a gene to the left (red) implies a higher correlation with EGF up-regulated genes in the reference platform, and the farther to the right (blue) implies a higher correlation with genes down-regulated upon EGF treatment in the reference platform. Studied gene sets correspond to lists of up- or down-regulated genes in each platform at 6 h of EGF treatment. Significantly enriched data sets are defined according to GSEA default settings (p

    Journal: BMC Genomics

    Article Title: Multiple platform assessment of the EGF dependent transcriptome by microarray and deep tag sequencing analysis

    doi: 10.1186/1471-2164-12-326

    Figure Lengend Snippet: GSEA analysis on significantly regulated gene sets across microarray platforms . Profile of the Running ES Score Positions of Gene Set Members on the Rank Ordered List using 6 h EGF treatment data according to each of the three microarray platforms. In each panel, the vertical black lines indicate the position of each of the genes of the tested gene set in the reference data set (ranked by average of the three respective EGF versus control log2ratios of replicate experiments). The green curve plots the ES (enrichment score), which is the running sum of the weighted enrichment score obtained from GSEA software. Within each queried gene set, the farther the position of a gene to the left (red) implies a higher correlation with EGF up-regulated genes in the reference platform, and the farther to the right (blue) implies a higher correlation with genes down-regulated upon EGF treatment in the reference platform. Studied gene sets correspond to lists of up- or down-regulated genes in each platform at 6 h of EGF treatment. Significantly enriched data sets are defined according to GSEA default settings (p

    Article Snippet: Here we use a combined approach to study the EGF dependent transcriptome of HeLa cells by using multiple long oligonucleotide based microarray platforms (from Agilent, Operon, and Illumina) in combination with digital gene expression profiling (DGE) with the Illumina Genome Analyzer.

    Techniques: Microarray, Software

    Microarray versus DGE analysis . (A) Overlap of unique and named genes shared among the 3 microarray platforms and genes detected by DGE. The pool of 14645 shared genes was used for further cross-platform analysis. The total numbers of genes for each platform and for all platforms combined are indicated. (B) Overlap of significantly regulated genes considering the 3 microarray platforms at 6 h after EGF treatment and the genes found regulated after assessing significance by grouping microarray and DGE data in a RankProd analysis. Left panels show up-regulated genes and right panels show down-regulated genes.

    Journal: BMC Genomics

    Article Title: Multiple platform assessment of the EGF dependent transcriptome by microarray and deep tag sequencing analysis

    doi: 10.1186/1471-2164-12-326

    Figure Lengend Snippet: Microarray versus DGE analysis . (A) Overlap of unique and named genes shared among the 3 microarray platforms and genes detected by DGE. The pool of 14645 shared genes was used for further cross-platform analysis. The total numbers of genes for each platform and for all platforms combined are indicated. (B) Overlap of significantly regulated genes considering the 3 microarray platforms at 6 h after EGF treatment and the genes found regulated after assessing significance by grouping microarray and DGE data in a RankProd analysis. Left panels show up-regulated genes and right panels show down-regulated genes.

    Article Snippet: Here we use a combined approach to study the EGF dependent transcriptome of HeLa cells by using multiple long oligonucleotide based microarray platforms (from Agilent, Operon, and Illumina) in combination with digital gene expression profiling (DGE) with the Illumina Genome Analyzer.

    Techniques: Microarray

    Correlation between microarrays and Illumina GA-I sequencing . (A) Comparison of estimated log2ratios from DGE ( Y -axis) and the mean of all microarray platforms ( X -axis). We consider only genes that were interrogated using all platforms and genes with a mean number of counts across lanes greater than 0. Genes with counts greater than 32 reads (colored red or green) or less than (black) 32 reads in at least one sample are shown. (Red dots) Genes called differentially expressed based on DGE data at an 10% FDR by RankProd. (Green dots) Genes not called as differentially expressed but above 32 counts. (Inset box) Correlation between technologies is higher when considering genes above the 32 count detection level (0.57) than when all genes are included (0.49). (B-C) Concordance at the top (CAT) plots of the different platforms with the 500 top genes from a reference platform, shown for Agilent in (B) and DGE in (C). See inset box for color codes identifying each platforms compared to the remaining platform used as reference. (D) Correlation plots with regression lines between log2ratios of the five high content platforms measurements (Y-axis) and quantitative real time PCR results using SYBR green assays (X-axis), based on measurements for 21 genes at the 6 h time point (see Additional file 2 , Table S1).

    Journal: BMC Genomics

    Article Title: Multiple platform assessment of the EGF dependent transcriptome by microarray and deep tag sequencing analysis

    doi: 10.1186/1471-2164-12-326

    Figure Lengend Snippet: Correlation between microarrays and Illumina GA-I sequencing . (A) Comparison of estimated log2ratios from DGE ( Y -axis) and the mean of all microarray platforms ( X -axis). We consider only genes that were interrogated using all platforms and genes with a mean number of counts across lanes greater than 0. Genes with counts greater than 32 reads (colored red or green) or less than (black) 32 reads in at least one sample are shown. (Red dots) Genes called differentially expressed based on DGE data at an 10% FDR by RankProd. (Green dots) Genes not called as differentially expressed but above 32 counts. (Inset box) Correlation between technologies is higher when considering genes above the 32 count detection level (0.57) than when all genes are included (0.49). (B-C) Concordance at the top (CAT) plots of the different platforms with the 500 top genes from a reference platform, shown for Agilent in (B) and DGE in (C). See inset box for color codes identifying each platforms compared to the remaining platform used as reference. (D) Correlation plots with regression lines between log2ratios of the five high content platforms measurements (Y-axis) and quantitative real time PCR results using SYBR green assays (X-axis), based on measurements for 21 genes at the 6 h time point (see Additional file 2 , Table S1).

    Article Snippet: Here we use a combined approach to study the EGF dependent transcriptome of HeLa cells by using multiple long oligonucleotide based microarray platforms (from Agilent, Operon, and Illumina) in combination with digital gene expression profiling (DGE) with the Illumina Genome Analyzer.

    Techniques: Sequencing, Microarray, Real-time Polymerase Chain Reaction, SYBR Green Assay

    Microarray interplatform analysis . (A) Overlap of unique and named genes shared among the 3 microarray platforms used in this study. The pool of 17070 shared genes was used for further cross-platform analysis. The total numbers of genes for each platform and for all platforms combined are indicated. (B) Overlap of significantly regulated genes at 6 h after EGF treatment considering each of the 3 microarray platforms independently.

    Journal: BMC Genomics

    Article Title: Multiple platform assessment of the EGF dependent transcriptome by microarray and deep tag sequencing analysis

    doi: 10.1186/1471-2164-12-326

    Figure Lengend Snippet: Microarray interplatform analysis . (A) Overlap of unique and named genes shared among the 3 microarray platforms used in this study. The pool of 17070 shared genes was used for further cross-platform analysis. The total numbers of genes for each platform and for all platforms combined are indicated. (B) Overlap of significantly regulated genes at 6 h after EGF treatment considering each of the 3 microarray platforms independently.

    Article Snippet: Here we use a combined approach to study the EGF dependent transcriptome of HeLa cells by using multiple long oligonucleotide based microarray platforms (from Agilent, Operon, and Illumina) in combination with digital gene expression profiling (DGE) with the Illumina Genome Analyzer.

    Techniques: Microarray