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  • 99
    New England Biolabs dna input
    Example of yield measurements obtained with the <t>NEBNext</t> kit when comparing the amount of <t>DNA</t> at each step versus the initial DNA input (500 ng). Blue bars correspond to values measured using the PhiX specific primers and reflect the DNA loss at each step mainly due to pipetting and bead clean up. The orange bar corresponds to the value measured with the adaptor specific primers and reflects the amount of DNA in the sample bearing adaptor after the ligation step in comparison with the initial DNA input. The green bar corresponds to the value measured with the P5/P7 primers and reflects the amount of sequencable DNA in the sample at the end of the library preparation
    Dna Input, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 99/100, based on 36 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Thermo Fisher control input dna
    Example of yield measurements obtained with the <t>NEBNext</t> kit when comparing the amount of <t>DNA</t> at each step versus the initial DNA input (500 ng). Blue bars correspond to values measured using the PhiX specific primers and reflect the DNA loss at each step mainly due to pipetting and bead clean up. The orange bar corresponds to the value measured with the adaptor specific primers and reflects the amount of DNA in the sample bearing adaptor after the ligation step in comparison with the initial DNA input. The green bar corresponds to the value measured with the P5/P7 primers and reflects the amount of sequencable DNA in the sample at the end of the library preparation
    Control Input Dna, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 16 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Millipore input dna
    The levels of H3K27me3 at promoter regions in GMPs. Visualization of ChIP-seq data of the H3K27me3 levels of several developmental regulator genes ( Hoxd locus, Gata4 , and Pax5 ), tumor suppressor genes ( Cdkn2a and Cdkn2b ) and oncogenes ( Hmga2 and Pbx3 ) in GMPs from WT, Tet2 KD/KD , Ezh2 Δ/Δ , and Tet2 KD/KD Ezh2 Δ/Δ mice at 4 mo after transplantation using the Integrative Genomics Viewer (IGV). Schematic diagram of these gene loci indicates their genomic structures. Exons and untranslated regions are demarcated by large and small black boxes, respectively. Data of ChIP analyses at the promoters of selected genes are also depicted. Quantitative ChIP analyses of GMPs from WT and Ezh2 Δ/Δ mice at 6 mo after transplantation were performed. The relative amounts of <t>immunoprecipitated</t> <t>DNA</t> are depicted as a percentage of input DNA. N.D. indicates not detected. The data are shown as the mean ± SEM for triplicate analyses. Regions amplified from the precipitated DNA by site-specific quantitative PCR are indicated by arrows. *, P
    Input Dna, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 1511 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    91
    Bio-Rad dna input
    Measuring translocations after HBB and AAVS1 di-genic targeting. ( a ) Schematic showing the HBB gene on chromosome 11 and the AAVS1 locus on chromosome 19. The Cas9 cut sites are shown in red. One of the two possible monocentric translocations is shown. ( b ) The reference sequence of the HBB-AAVS1 translocation is shown in the top. Below are representative translocation sequences from GFP - BFP - HSPCs sorted seven days after targeting (see Figure 3e , left panel). ( c ) Representative ddPCR analyses quantifying translocations in NTC (non-template control), mock-electroporated, and GFP - BFP - cells (see Figure 3e , right panel). The reference assay quantifies <t>TERT</t> gene copies used to normalize for <t>DNA</t> input. The translocation assay probe binds 50 bp away from the junction and none of the identified translocations would therefore exclude probe binding.
    Dna Input, supplied by Bio-Rad, used in various techniques. Bioz Stars score: 91/100, based on 23 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    91
    Illumina Inc dna input
    Coverage of the microbial genomes, Olsenella profusa (62% GC) (left) and Bacillus megaterium (38% GC) (right) with (A) <t>MGIEasy</t> PCR-Free <t>DNA</t> Library Pre Set and (B) MGIEasy FS PCR-Free DNA Library Pre Set. Read coverage across the range of the GC content, calculated in 100 bp windows (pink bars) and normalized coverage (colored dots). 3 replicates (red, blue and green dots) were included in the normalized coverage VS GC content analysis.
    Dna Input, supplied by Illumina Inc, used in various techniques. Bioz Stars score: 91/100, based on 52 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    ATUM input dna
    ChIP analysis of DMR1 occupancy by <t>Ctcf,</t> Parp1, PARs and Dnmt1 ( A ) Schematic map of the locus. The DMR1 region is expanded to show the approximate position of putative Ctcf-binding sites and of PCR primers used to detect the presence of specific <t>DNA</t> sequences in ChIP complexes. Circles represent CpG dinuclotides. H, HpaII sites. ( B ) ChIP analysis of DMR1 region carried out with anti-Ctcf, anti-Parp1, anti-Dnmt1 and anti-PAR Abs. Beads alone (No Ab) and anti-IgG Abs were used as negative controls. Real-time PCR data are expressed as percentage of the signal detected for the non-immunoprecipitated input (4% of the chromatin subjected to immunoprecipitation) taken as 100%. The Actb promoter served as a control. ( C ) Standard ChIP assay for Ctcf was followed by RE-ChIP to assess the co-occupancy of Ctcf, with Parp1, PARs and Dnmt1 at the Ctcf DNA target sites within the DMR1. Real-time PCR was performed for the DMR1 b and c fragments. The efficiency of RE-ChIP at each of the sites was calculated as a percentage of the chromatin input that co-purified with Ctcf in the first ChIP (10% of the Ctcf ChIP fraction). Results are means±S.E.M. calculated from three experiments. * P
    Input Dna, supplied by ATUM, used in various techniques. Bioz Stars score: 92/100, based on 57 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    94
    New England Biolabs input dna
    Evenness of coverage for B. anthracis (a) and E. coli (b) genome sequencing using <t>NEBNext</t> library preparation with 100 ng <t>DNA</t> input (top graph) and TruSeq library preparation with 1,000 ng DNA input (bottom graph). Plots are normalized by the average coverage in each figure.
    Input Dna, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 94/100, based on 909 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Thermo Fisher dna ladder input
    Evenness of coverage for B. anthracis (a) and E. coli (b) genome sequencing using <t>NEBNext</t> library preparation with 100 ng <t>DNA</t> input (top graph) and TruSeq library preparation with 1,000 ng DNA input (bottom graph). Plots are normalized by the average coverage in each figure.
    Dna Ladder Input, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 92/100, based on 10 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Average 92 stars, based on 10 article reviews
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    93
    Pacific Biosciences low input dna
    Evenness of coverage for B. anthracis (a) and E. coli (b) genome sequencing using <t>NEBNext</t> library preparation with 100 ng <t>DNA</t> input (top graph) and TruSeq library preparation with 1,000 ng DNA input (bottom graph). Plots are normalized by the average coverage in each figure.
    Low Input Dna, supplied by Pacific Biosciences, used in various techniques. Bioz Stars score: 93/100, based on 18 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    91
    Illumina Inc input dna input
    Evenness of coverage for B. anthracis (a) and E. coli (b) genome sequencing using <t>NEBNext</t> library preparation with 100 ng <t>DNA</t> input (top graph) and TruSeq library preparation with 1,000 ng DNA input (bottom graph). Plots are normalized by the average coverage in each figure.
    Input Dna Input, supplied by Illumina Inc, used in various techniques. Bioz Stars score: 91/100, based on 4 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Average 91 stars, based on 4 article reviews
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    85
    Roche input dna input
    Evenness of coverage for B. anthracis (a) and E. coli (b) genome sequencing using <t>NEBNext</t> library preparation with 100 ng <t>DNA</t> input (top graph) and TruSeq library preparation with 1,000 ng DNA input (bottom graph). Plots are normalized by the average coverage in each figure.
    Input Dna Input, supplied by Roche, used in various techniques. Bioz Stars score: 85/100, based on 8 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Average 85 stars, based on 8 article reviews
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    Image Search Results


    Example of yield measurements obtained with the NEBNext kit when comparing the amount of DNA at each step versus the initial DNA input (500 ng). Blue bars correspond to values measured using the PhiX specific primers and reflect the DNA loss at each step mainly due to pipetting and bead clean up. The orange bar corresponds to the value measured with the adaptor specific primers and reflects the amount of DNA in the sample bearing adaptor after the ligation step in comparison with the initial DNA input. The green bar corresponds to the value measured with the P5/P7 primers and reflects the amount of sequencable DNA in the sample at the end of the library preparation

    Journal: BMC Genomics

    Article Title: Quantitation of next generation sequencing library preparation protocol efficiencies using droplet digital PCR assays - a systematic comparison of DNA library preparation kits for Illumina sequencing

    doi: 10.1186/s12864-016-2757-4

    Figure Lengend Snippet: Example of yield measurements obtained with the NEBNext kit when comparing the amount of DNA at each step versus the initial DNA input (500 ng). Blue bars correspond to values measured using the PhiX specific primers and reflect the DNA loss at each step mainly due to pipetting and bead clean up. The orange bar corresponds to the value measured with the adaptor specific primers and reflects the amount of DNA in the sample bearing adaptor after the ligation step in comparison with the initial DNA input. The green bar corresponds to the value measured with the P5/P7 primers and reflects the amount of sequencable DNA in the sample at the end of the library preparation

    Article Snippet: Certain kits are specifically designed for low DNA input such as the NEBNext Ultra and Swift Accel-NGS 2S, while others such as the KAPA ones accept a wide range of DNA input from a 1 ng to 1 μg.

    Techniques: Ligation

    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: It should be noted that kits are now available that offer the ligation addition of Illumina adapters with starting DNA inputs as little as 5 ng (NEBNext Ultra, NEB).

    Techniques: Amplification, Polymerase Chain Reaction, Ligation, Sequencing

    Evenness of coverage for B. anthracis (a) and E. coli (b) genome sequencing using NEBNext library preparation with 100 ng DNA input (top graph) and TruSeq library preparation with 1,000 ng DNA input (bottom graph). Plots are normalized by the average coverage in each figure.

    Journal: International Journal of Genomics

    Article Title: Facile, High Quality Sequencing of Bacterial Genomes from Small Amounts of DNA

    doi: 10.1155/2014/434575

    Figure Lengend Snippet: Evenness of coverage for B. anthracis (a) and E. coli (b) genome sequencing using NEBNext library preparation with 100 ng DNA input (top graph) and TruSeq library preparation with 1,000 ng DNA input (bottom graph). Plots are normalized by the average coverage in each figure.

    Article Snippet: Standard input DNA amounts were 100 ng, approximately 10x lower than the required amount for the Illumina TruSeq kit and 10x higher than the minimum DNA inputs per NEBNext Ultra manual specifications.

    Techniques: Sequencing

    Evenness of coverage plots for B. thailandensis A ((a) is chromosome 1 and (b) is chromosome 2) and B. thailandensis B ((c) is chromosome 1 and (d) is chromosome 2) genome sequencing. Top graph within each panel shows NEBNext library preparation data with 10 ng or 100 ng DNA input (black color is for 10 ng samples and red color is for 100 ng samples). Bottom graph within each panel shows TruSeq library preparation data with 1,000 ng DNA input. Plots are normalized by the average coverage in each figure.

    Journal: International Journal of Genomics

    Article Title: Facile, High Quality Sequencing of Bacterial Genomes from Small Amounts of DNA

    doi: 10.1155/2014/434575

    Figure Lengend Snippet: Evenness of coverage plots for B. thailandensis A ((a) is chromosome 1 and (b) is chromosome 2) and B. thailandensis B ((c) is chromosome 1 and (d) is chromosome 2) genome sequencing. Top graph within each panel shows NEBNext library preparation data with 10 ng or 100 ng DNA input (black color is for 10 ng samples and red color is for 100 ng samples). Bottom graph within each panel shows TruSeq library preparation data with 1,000 ng DNA input. Plots are normalized by the average coverage in each figure.

    Article Snippet: Standard input DNA amounts were 100 ng, approximately 10x lower than the required amount for the Illumina TruSeq kit and 10x higher than the minimum DNA inputs per NEBNext Ultra manual specifications.

    Techniques: Sequencing

    The levels of H3K27me3 at promoter regions in GMPs. Visualization of ChIP-seq data of the H3K27me3 levels of several developmental regulator genes ( Hoxd locus, Gata4 , and Pax5 ), tumor suppressor genes ( Cdkn2a and Cdkn2b ) and oncogenes ( Hmga2 and Pbx3 ) in GMPs from WT, Tet2 KD/KD , Ezh2 Δ/Δ , and Tet2 KD/KD Ezh2 Δ/Δ mice at 4 mo after transplantation using the Integrative Genomics Viewer (IGV). Schematic diagram of these gene loci indicates their genomic structures. Exons and untranslated regions are demarcated by large and small black boxes, respectively. Data of ChIP analyses at the promoters of selected genes are also depicted. Quantitative ChIP analyses of GMPs from WT and Ezh2 Δ/Δ mice at 6 mo after transplantation were performed. The relative amounts of immunoprecipitated DNA are depicted as a percentage of input DNA. N.D. indicates not detected. The data are shown as the mean ± SEM for triplicate analyses. Regions amplified from the precipitated DNA by site-specific quantitative PCR are indicated by arrows. *, P

    Journal: The Journal of Experimental Medicine

    Article Title: Concurrent loss of Ezh2 and Tet2 cooperates in the pathogenesis of myelodysplastic disorders

    doi: 10.1084/jem.20131144

    Figure Lengend Snippet: The levels of H3K27me3 at promoter regions in GMPs. Visualization of ChIP-seq data of the H3K27me3 levels of several developmental regulator genes ( Hoxd locus, Gata4 , and Pax5 ), tumor suppressor genes ( Cdkn2a and Cdkn2b ) and oncogenes ( Hmga2 and Pbx3 ) in GMPs from WT, Tet2 KD/KD , Ezh2 Δ/Δ , and Tet2 KD/KD Ezh2 Δ/Δ mice at 4 mo after transplantation using the Integrative Genomics Viewer (IGV). Schematic diagram of these gene loci indicates their genomic structures. Exons and untranslated regions are demarcated by large and small black boxes, respectively. Data of ChIP analyses at the promoters of selected genes are also depicted. Quantitative ChIP analyses of GMPs from WT and Ezh2 Δ/Δ mice at 6 mo after transplantation were performed. The relative amounts of immunoprecipitated DNA are depicted as a percentage of input DNA. N.D. indicates not detected. The data are shown as the mean ± SEM for triplicate analyses. Regions amplified from the precipitated DNA by site-specific quantitative PCR are indicated by arrows. *, P

    Article Snippet: Immunoprecipitated DNA and input DNA were treated with RNase A (Sigma-Aldrich) and proteinase K (Roche), and purified with a QIAquick PCR purification kit (QIAGEN).

    Techniques: Chromatin Immunoprecipitation, Mouse Assay, Transplantation Assay, Immunoprecipitation, Amplification, Real-time Polymerase Chain Reaction

    Assessment of the specificity of the ChIP protocol. A) Arx immunoprecipitation from N2a cells transfected with an Arx-expressing vector or a vector alone and using the Arx C-14 antibody from Santa Cruz. Detection using the anti-Arx-HD antibody revealed a major band at approximately 80 kD, which corresponds to the size of Arx protein. TE: total extracts, IP: immunoprecipitation, Ig: immunoglobulins. B) Enrichment of Ebf3 , Lmo1 and Shox2 promoter regions was assessed by qPCR using either Arx or PolII-immunoprecipitates or using no antibody and was compared to the total input. Gapdh was used as a positive control with DNA Pol II. Here, we show an example of the results obtained in a ChIP experiment with Arx C-14 antibody from Santa Cruz. The enrichment of Ebf3 , Lmo1 and Shox2 was checked similarly for each replicate experiment before DNA was applied to the microarrays.

    Journal: PLoS ONE

    Article Title: High-Throughput Analysis of Promoter Occupancy Reveals New Targets for Arx, a Gene Mutated in Mental Retardation and Interneuronopathies

    doi: 10.1371/journal.pone.0025181

    Figure Lengend Snippet: Assessment of the specificity of the ChIP protocol. A) Arx immunoprecipitation from N2a cells transfected with an Arx-expressing vector or a vector alone and using the Arx C-14 antibody from Santa Cruz. Detection using the anti-Arx-HD antibody revealed a major band at approximately 80 kD, which corresponds to the size of Arx protein. TE: total extracts, IP: immunoprecipitation, Ig: immunoglobulins. B) Enrichment of Ebf3 , Lmo1 and Shox2 promoter regions was assessed by qPCR using either Arx or PolII-immunoprecipitates or using no antibody and was compared to the total input. Gapdh was used as a positive control with DNA Pol II. Here, we show an example of the results obtained in a ChIP experiment with Arx C-14 antibody from Santa Cruz. The enrichment of Ebf3 , Lmo1 and Shox2 was checked similarly for each replicate experiment before DNA was applied to the microarrays.

    Article Snippet: DNA amplification, labelling and hybridization on microarrays Equivalent amounts of immunoprecipitated chromatin and total input DNA were amplified in parallel, using a random primer method with the GenomePlex Complete Whole Genome Amplification Kit (15 cycles), according to the manufacturer's instructions (Sigma).

    Techniques: Chromatin Immunoprecipitation, Immunoprecipitation, Transfection, Expressing, Plasmid Preparation, Real-time Polymerase Chain Reaction, Positive Control

    ChIP-chip results obtained from mouse embryonic brain. A) Graph representing log 2 probe intensities of Arx-immunoprecipitated DNA (IP) and input DNA obtained in a representative ChIP experiment. The red dots indicate the probes enriched in Arx-immunoprecipitates compared to total input DNA. B) Example of the enrichment profiles of Arx-bound promoter regions visualized by DNA analytics. The 3 lines represent data obtained from 3 independent ChIP-chip experiments and show the reproducibility between the 3 replicates. Contrarily to N2a cells, in which several continuous probes were often found enriched, only one or two probes were found enriched per gene. C) Venn diagram illustrating the overlap (black) between Arx-immunoprecipitated genes in transfected N2a cells (blue) and mouse embryonic brain (red), and the number of genes with at least 75% match to Arx-binding motif.

    Journal: PLoS ONE

    Article Title: High-Throughput Analysis of Promoter Occupancy Reveals New Targets for Arx, a Gene Mutated in Mental Retardation and Interneuronopathies

    doi: 10.1371/journal.pone.0025181

    Figure Lengend Snippet: ChIP-chip results obtained from mouse embryonic brain. A) Graph representing log 2 probe intensities of Arx-immunoprecipitated DNA (IP) and input DNA obtained in a representative ChIP experiment. The red dots indicate the probes enriched in Arx-immunoprecipitates compared to total input DNA. B) Example of the enrichment profiles of Arx-bound promoter regions visualized by DNA analytics. The 3 lines represent data obtained from 3 independent ChIP-chip experiments and show the reproducibility between the 3 replicates. Contrarily to N2a cells, in which several continuous probes were often found enriched, only one or two probes were found enriched per gene. C) Venn diagram illustrating the overlap (black) between Arx-immunoprecipitated genes in transfected N2a cells (blue) and mouse embryonic brain (red), and the number of genes with at least 75% match to Arx-binding motif.

    Article Snippet: DNA amplification, labelling and hybridization on microarrays Equivalent amounts of immunoprecipitated chromatin and total input DNA were amplified in parallel, using a random primer method with the GenomePlex Complete Whole Genome Amplification Kit (15 cycles), according to the manufacturer's instructions (Sigma).

    Techniques: Chromatin Immunoprecipitation, Immunoprecipitation, Transfection, Binding Assay

    ChIP-chip results obtained from Arx-transfected N2a cells. A) Graph representing log 2 probe intensities of Arx-immunoprecipitated DNA (IP) and input DNA obtained in a representative ChIP experiment. The red dots indicate the probes enriched in Arx-immunoprecipitates compared to total input DNA. B) Examples of the enrichment profiles of Arx-bound promoter regions visualized by DNA analytics software. The 3 lines represent data obtained from 3 independent ChIP-chip experiments and show the reproducibility between the 3 replicates. C) The resulting weighted matrix discovered through the MDModule analysis (top) appears to be similar to the motif identified by Berger et al. [27] (bottom). D) Frequency distribution of scores. The TAATTA motif identified by MDModule was significantly more present in ChIP-enriched sequences (red curve) by comparison to negative control sequences (blue curve).

    Journal: PLoS ONE

    Article Title: High-Throughput Analysis of Promoter Occupancy Reveals New Targets for Arx, a Gene Mutated in Mental Retardation and Interneuronopathies

    doi: 10.1371/journal.pone.0025181

    Figure Lengend Snippet: ChIP-chip results obtained from Arx-transfected N2a cells. A) Graph representing log 2 probe intensities of Arx-immunoprecipitated DNA (IP) and input DNA obtained in a representative ChIP experiment. The red dots indicate the probes enriched in Arx-immunoprecipitates compared to total input DNA. B) Examples of the enrichment profiles of Arx-bound promoter regions visualized by DNA analytics software. The 3 lines represent data obtained from 3 independent ChIP-chip experiments and show the reproducibility between the 3 replicates. C) The resulting weighted matrix discovered through the MDModule analysis (top) appears to be similar to the motif identified by Berger et al. [27] (bottom). D) Frequency distribution of scores. The TAATTA motif identified by MDModule was significantly more present in ChIP-enriched sequences (red curve) by comparison to negative control sequences (blue curve).

    Article Snippet: DNA amplification, labelling and hybridization on microarrays Equivalent amounts of immunoprecipitated chromatin and total input DNA were amplified in parallel, using a random primer method with the GenomePlex Complete Whole Genome Amplification Kit (15 cycles), according to the manufacturer's instructions (Sigma).

    Techniques: Chromatin Immunoprecipitation, Transfection, Immunoprecipitation, Software, Negative Control

    Confirmation of Arx binding to candidate promoter regions. A) Arx-immunoprecipitated DNA was compared to input DNA by ChIP/QFM-PCR to determine ChIP enrichment of 21 putative target genes. No enrichment occurred for the Vapb promoter which was consistently negative in all ChIP experiments. Bar heights represent log 10 enrichment of the signal obtained for Arx-immunoprecipitated DNA versus input DNA for each promoter using site-specific primers. The arrows above the bars indicate sequences in which the previously defined Arx-binding motif was found. B) Confirmation of Arx binding to 5 different promoter sequences identified by ChIP using a luciferase reporter gene assay. Firefly luciferase data were normalized to Renilla luciferase expression and data are presented as the percentage of transcriptional activity compared to the vector control. Arx regulation was confirmed for Lmo1 , Lmo3 , Sh3tc2 , Calb2 and Cdh2 promoter regions as transcriptional activity was repressed in the presence of Arx by comparison to the control transfection, whereas it had no effect on the plasmid alone pGL4.23. Error bars indicate SEM.

    Journal: PLoS ONE

    Article Title: High-Throughput Analysis of Promoter Occupancy Reveals New Targets for Arx, a Gene Mutated in Mental Retardation and Interneuronopathies

    doi: 10.1371/journal.pone.0025181

    Figure Lengend Snippet: Confirmation of Arx binding to candidate promoter regions. A) Arx-immunoprecipitated DNA was compared to input DNA by ChIP/QFM-PCR to determine ChIP enrichment of 21 putative target genes. No enrichment occurred for the Vapb promoter which was consistently negative in all ChIP experiments. Bar heights represent log 10 enrichment of the signal obtained for Arx-immunoprecipitated DNA versus input DNA for each promoter using site-specific primers. The arrows above the bars indicate sequences in which the previously defined Arx-binding motif was found. B) Confirmation of Arx binding to 5 different promoter sequences identified by ChIP using a luciferase reporter gene assay. Firefly luciferase data were normalized to Renilla luciferase expression and data are presented as the percentage of transcriptional activity compared to the vector control. Arx regulation was confirmed for Lmo1 , Lmo3 , Sh3tc2 , Calb2 and Cdh2 promoter regions as transcriptional activity was repressed in the presence of Arx by comparison to the control transfection, whereas it had no effect on the plasmid alone pGL4.23. Error bars indicate SEM.

    Article Snippet: DNA amplification, labelling and hybridization on microarrays Equivalent amounts of immunoprecipitated chromatin and total input DNA were amplified in parallel, using a random primer method with the GenomePlex Complete Whole Genome Amplification Kit (15 cycles), according to the manufacturer's instructions (Sigma).

    Techniques: Binding Assay, Immunoprecipitation, Chromatin Immunoprecipitation, Polymerase Chain Reaction, Luciferase, Reporter Gene Assay, Expressing, Activity Assay, Plasmid Preparation, Transfection

    Comparison of methylation data between MeDIP/NimbleGen Promoter + CpGi Array and Sequenom MassArray: Stk31 CpGi promoter region . The Stk31 gene region with a CpGi promoter region that is methylated in the liver, but not in ES or testis cells. The box indicates the position of the methylation peak in the liver that overlaps with the CpGi promoter region analyzed by Sequenom. In the epigram of Sequenom methylation analysis shown at the bottom panel, the blue circles indicate 100% methylation, green circles indicate around 50%methylation and yellow circles indicate 0% methylation. The results from biological duplicate samples are shown. The bottom lane (Sequenom Methylation Analysis) shows the results of analysis of testis DNA after in vitro methylation using M. Sss1 methylase (New England Biolabs).

    Journal: BMC Genomics

    Article Title: Genome-wide survey reveals dynamic widespread tissue-specific changes in DNA methylation during development

    doi: 10.1186/1471-2164-12-231

    Figure Lengend Snippet: Comparison of methylation data between MeDIP/NimbleGen Promoter + CpGi Array and Sequenom MassArray: Stk31 CpGi promoter region . The Stk31 gene region with a CpGi promoter region that is methylated in the liver, but not in ES or testis cells. The box indicates the position of the methylation peak in the liver that overlaps with the CpGi promoter region analyzed by Sequenom. In the epigram of Sequenom methylation analysis shown at the bottom panel, the blue circles indicate 100% methylation, green circles indicate around 50%methylation and yellow circles indicate 0% methylation. The results from biological duplicate samples are shown. The bottom lane (Sequenom Methylation Analysis) shows the results of analysis of testis DNA after in vitro methylation using M. Sss1 methylase (New England Biolabs).

    Article Snippet: After validating the enrichment of MeDIP DNA, MeDIP DNA and control input DNA were amplified by whole-genome amplification kit (Sigma Aldrich), followed by purification (QIAGEN Quick PCR Purification Kit) and then sent to NimbleGen for Microarray hybridization according to their standard protocol for the array.

    Techniques: Methylation, Methylated DNA Immunoprecipitation, In Vitro

    Measuring translocations after HBB and AAVS1 di-genic targeting. ( a ) Schematic showing the HBB gene on chromosome 11 and the AAVS1 locus on chromosome 19. The Cas9 cut sites are shown in red. One of the two possible monocentric translocations is shown. ( b ) The reference sequence of the HBB-AAVS1 translocation is shown in the top. Below are representative translocation sequences from GFP - BFP - HSPCs sorted seven days after targeting (see Figure 3e , left panel). ( c ) Representative ddPCR analyses quantifying translocations in NTC (non-template control), mock-electroporated, and GFP - BFP - cells (see Figure 3e , right panel). The reference assay quantifies TERT gene copies used to normalize for DNA input. The translocation assay probe binds 50 bp away from the junction and none of the identified translocations would therefore exclude probe binding.

    Journal: eLife

    Article Title: Multiplexed genetic engineering of human hematopoietic stem and progenitor cells using CRISPR/Cas9 and AAV6

    doi: 10.7554/eLife.27873

    Figure Lengend Snippet: Measuring translocations after HBB and AAVS1 di-genic targeting. ( a ) Schematic showing the HBB gene on chromosome 11 and the AAVS1 locus on chromosome 19. The Cas9 cut sites are shown in red. One of the two possible monocentric translocations is shown. ( b ) The reference sequence of the HBB-AAVS1 translocation is shown in the top. Below are representative translocation sequences from GFP - BFP - HSPCs sorted seven days after targeting (see Figure 3e , left panel). ( c ) Representative ddPCR analyses quantifying translocations in NTC (non-template control), mock-electroporated, and GFP - BFP - cells (see Figure 3e , right panel). The reference assay quantifies TERT gene copies used to normalize for DNA input. The translocation assay probe binds 50 bp away from the junction and none of the identified translocations would therefore exclude probe binding.

    Article Snippet: A HEX reference assay detecting copy number input of the TERT gene was used to normalize for genomic DNA input (Bio-Rad: saCP1000100).

    Techniques: Sequencing, Translocation Assay, Binding Assay

    Coverage of the microbial genomes, Olsenella profusa (62% GC) (left) and Bacillus megaterium (38% GC) (right) with (A) MGIEasy PCR-Free DNA Library Pre Set and (B) MGIEasy FS PCR-Free DNA Library Pre Set. Read coverage across the range of the GC content, calculated in 100 bp windows (pink bars) and normalized coverage (colored dots). 3 replicates (red, blue and green dots) were included in the normalized coverage VS GC content analysis.

    Journal: bioRxiv

    Article Title: Advanced Whole Genome Sequencing Using a Complete PCR-free Massively Parallel Sequencing (MPS) Workflow

    doi: 10.1101/2019.12.20.885517

    Figure Lengend Snippet: Coverage of the microbial genomes, Olsenella profusa (62% GC) (left) and Bacillus megaterium (38% GC) (right) with (A) MGIEasy PCR-Free DNA Library Pre Set and (B) MGIEasy FS PCR-Free DNA Library Pre Set. Read coverage across the range of the GC content, calculated in 100 bp windows (pink bars) and normalized coverage (colored dots). 3 replicates (red, blue and green dots) were included in the normalized coverage VS GC content analysis.

    Article Snippet: It should be noted that MGIEasy kit prepared libraries used 1 μg or 250ng DNA input, far less than the input amount of datasets downloaded from Illumina Basespace website.

    Techniques: Polymerase Chain Reaction

    DNBSEQTM WGS PCR vs PCR-free workflows and general performance of PCR-free libraries. (a) MPS library construction workflows of WGS PCR and PCR-free libraries. RCA ( r olling c ircle a mplification) is used to increase signal intensity during array formation, which is followed by sequencing DNBs with DNBSEQ TM technology. Individual copies from the same DNB are replicated independently using the same ssCir template. Therefore, amplification errors cannot accumulate. Black, genomic DNA; Gray rectangle, adapter; green, barcode; red, amplification errors. (b) Two sets of 9 replicates from 1 μg NA12878 DNA were processed with MGIEasy PCR-Free DNA Library Prep Set (blue) or MGIEasy FS PCR-Free DNA Library Prep Set (orange). The GC content, Duplication rate, Median Insert size, and regions with > 10x Coverage were calculated and plotted. The error bars represent the standard deviations across the replicates.

    Journal: bioRxiv

    Article Title: Advanced Whole Genome Sequencing Using a Complete PCR-free Massively Parallel Sequencing (MPS) Workflow

    doi: 10.1101/2019.12.20.885517

    Figure Lengend Snippet: DNBSEQTM WGS PCR vs PCR-free workflows and general performance of PCR-free libraries. (a) MPS library construction workflows of WGS PCR and PCR-free libraries. RCA ( r olling c ircle a mplification) is used to increase signal intensity during array formation, which is followed by sequencing DNBs with DNBSEQ TM technology. Individual copies from the same DNB are replicated independently using the same ssCir template. Therefore, amplification errors cannot accumulate. Black, genomic DNA; Gray rectangle, adapter; green, barcode; red, amplification errors. (b) Two sets of 9 replicates from 1 μg NA12878 DNA were processed with MGIEasy PCR-Free DNA Library Prep Set (blue) or MGIEasy FS PCR-Free DNA Library Prep Set (orange). The GC content, Duplication rate, Median Insert size, and regions with > 10x Coverage were calculated and plotted. The error bars represent the standard deviations across the replicates.

    Article Snippet: It should be noted that MGIEasy kit prepared libraries used 1 μg or 250ng DNA input, far less than the input amount of datasets downloaded from Illumina Basespace website.

    Techniques: Polymerase Chain Reaction, Sequencing, Amplification

    ChIP analysis of DMR1 occupancy by Ctcf, Parp1, PARs and Dnmt1 ( A ) Schematic map of the locus. The DMR1 region is expanded to show the approximate position of putative Ctcf-binding sites and of PCR primers used to detect the presence of specific DNA sequences in ChIP complexes. Circles represent CpG dinuclotides. H, HpaII sites. ( B ) ChIP analysis of DMR1 region carried out with anti-Ctcf, anti-Parp1, anti-Dnmt1 and anti-PAR Abs. Beads alone (No Ab) and anti-IgG Abs were used as negative controls. Real-time PCR data are expressed as percentage of the signal detected for the non-immunoprecipitated input (4% of the chromatin subjected to immunoprecipitation) taken as 100%. The Actb promoter served as a control. ( C ) Standard ChIP assay for Ctcf was followed by RE-ChIP to assess the co-occupancy of Ctcf, with Parp1, PARs and Dnmt1 at the Ctcf DNA target sites within the DMR1. Real-time PCR was performed for the DMR1 b and c fragments. The efficiency of RE-ChIP at each of the sites was calculated as a percentage of the chromatin input that co-purified with Ctcf in the first ChIP (10% of the Ctcf ChIP fraction). Results are means±S.E.M. calculated from three experiments. * P

    Journal: Biochemical Journal

    Article Title: ADP-ribose polymers localized on Ctcf-Parp1-Dnmt1 complex prevent methylation of Ctcf target sites

    doi: 10.1042/BJ20111417

    Figure Lengend Snippet: ChIP analysis of DMR1 occupancy by Ctcf, Parp1, PARs and Dnmt1 ( A ) Schematic map of the locus. The DMR1 region is expanded to show the approximate position of putative Ctcf-binding sites and of PCR primers used to detect the presence of specific DNA sequences in ChIP complexes. Circles represent CpG dinuclotides. H, HpaII sites. ( B ) ChIP analysis of DMR1 region carried out with anti-Ctcf, anti-Parp1, anti-Dnmt1 and anti-PAR Abs. Beads alone (No Ab) and anti-IgG Abs were used as negative controls. Real-time PCR data are expressed as percentage of the signal detected for the non-immunoprecipitated input (4% of the chromatin subjected to immunoprecipitation) taken as 100%. The Actb promoter served as a control. ( C ) Standard ChIP assay for Ctcf was followed by RE-ChIP to assess the co-occupancy of Ctcf, with Parp1, PARs and Dnmt1 at the Ctcf DNA target sites within the DMR1. Real-time PCR was performed for the DMR1 b and c fragments. The efficiency of RE-ChIP at each of the sites was calculated as a percentage of the chromatin input that co-purified with Ctcf in the first ChIP (10% of the Ctcf ChIP fraction). Results are means±S.E.M. calculated from three experiments. * P

    Article Snippet: ChIP-CHOP assay For ChIP-CHOP analysis of the DMR1 b and c fragments, the Ctcf-, Parp1- and Dnmt-pulled down DNA (20 μl), and input DNA (20 μl after 1:100 and 1:200 dilutions) from ChIP assays were digested for 1 h at 37°C with 10 units of either the methylation-sensitive HpaII restriction enzyme or its methylation-insensitive isoschizomer MspI (New England Biolabs).

    Techniques: Chromatin Immunoprecipitation, Binding Assay, Polymerase Chain Reaction, Real-time Polymerase Chain Reaction, Immunoprecipitation, Purification

    Ctcf, Parp1, and Dnmt1 bind to non-methylated DNA molecules within the DMR1 Ctcf, Parp1, and Dnmt1 ChIPs were performed for the DMR1 b and c fragments. End-point PCR was performed after digestion of ChIP fractions DNA and inputs with either HpaII (H, methylation sensitive) or MspI (M, methylation insensitive) following heat inactivation of the restriction enzymes. The input (4% of the chromatin subjected to immunoprecipitation) was diluted 1/100 or 1/200 before restriction. The uncut (U) fractions consisted in HpaII digestions preventively blocked by heat inactivation. W, PCR performed in the absence of added template.

    Journal: Biochemical Journal

    Article Title: ADP-ribose polymers localized on Ctcf-Parp1-Dnmt1 complex prevent methylation of Ctcf target sites

    doi: 10.1042/BJ20111417

    Figure Lengend Snippet: Ctcf, Parp1, and Dnmt1 bind to non-methylated DNA molecules within the DMR1 Ctcf, Parp1, and Dnmt1 ChIPs were performed for the DMR1 b and c fragments. End-point PCR was performed after digestion of ChIP fractions DNA and inputs with either HpaII (H, methylation sensitive) or MspI (M, methylation insensitive) following heat inactivation of the restriction enzymes. The input (4% of the chromatin subjected to immunoprecipitation) was diluted 1/100 or 1/200 before restriction. The uncut (U) fractions consisted in HpaII digestions preventively blocked by heat inactivation. W, PCR performed in the absence of added template.

    Article Snippet: ChIP-CHOP assay For ChIP-CHOP analysis of the DMR1 b and c fragments, the Ctcf-, Parp1- and Dnmt-pulled down DNA (20 μl), and input DNA (20 μl after 1:100 and 1:200 dilutions) from ChIP assays were digested for 1 h at 37°C with 10 units of either the methylation-sensitive HpaII restriction enzyme or its methylation-insensitive isoschizomer MspI (New England Biolabs).

    Techniques: Methylation, Polymerase Chain Reaction, Chromatin Immunoprecipitation, Immunoprecipitation

    Evenness of coverage for B. anthracis (a) and E. coli (b) genome sequencing using NEBNext library preparation with 100 ng DNA input (top graph) and TruSeq library preparation with 1,000 ng DNA input (bottom graph). Plots are normalized by the average coverage in each figure.

    Journal: International Journal of Genomics

    Article Title: Facile, High Quality Sequencing of Bacterial Genomes from Small Amounts of DNA

    doi: 10.1155/2014/434575

    Figure Lengend Snippet: Evenness of coverage for B. anthracis (a) and E. coli (b) genome sequencing using NEBNext library preparation with 100 ng DNA input (top graph) and TruSeq library preparation with 1,000 ng DNA input (bottom graph). Plots are normalized by the average coverage in each figure.

    Article Snippet: Several library preparation kits that require 1–100 ng of input DNA are now available (New England Biolabs' NEBNext, Illumina's TruSeq Nano, Bioo Scientific's NEXTflex, NuGEN's Ovation Ultralow, etc.).

    Techniques: Sequencing

    Evenness of coverage plots for B. thailandensis A ((a) is chromosome 1 and (b) is chromosome 2) and B. thailandensis B ((c) is chromosome 1 and (d) is chromosome 2) genome sequencing. Top graph within each panel shows NEBNext library preparation data with 10 ng or 100 ng DNA input (black color is for 10 ng samples and red color is for 100 ng samples). Bottom graph within each panel shows TruSeq library preparation data with 1,000 ng DNA input. Plots are normalized by the average coverage in each figure.

    Journal: International Journal of Genomics

    Article Title: Facile, High Quality Sequencing of Bacterial Genomes from Small Amounts of DNA

    doi: 10.1155/2014/434575

    Figure Lengend Snippet: Evenness of coverage plots for B. thailandensis A ((a) is chromosome 1 and (b) is chromosome 2) and B. thailandensis B ((c) is chromosome 1 and (d) is chromosome 2) genome sequencing. Top graph within each panel shows NEBNext library preparation data with 10 ng or 100 ng DNA input (black color is for 10 ng samples and red color is for 100 ng samples). Bottom graph within each panel shows TruSeq library preparation data with 1,000 ng DNA input. Plots are normalized by the average coverage in each figure.

    Article Snippet: Several library preparation kits that require 1–100 ng of input DNA are now available (New England Biolabs' NEBNext, Illumina's TruSeq Nano, Bioo Scientific's NEXTflex, NuGEN's Ovation Ultralow, etc.).

    Techniques: Sequencing