amplicons  (New England Biolabs)


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    Name:
    Quick Blunting Kit
    Description:
    Quick Blunting Kit 100 rxns
    Catalog Number:
    e1201l
    Price:
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    Size:
    100 rxns
    Category:
    Blunt end DNA Kits
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    Structured Review

    New England Biolabs amplicons
    Quick Blunting Kit
    Quick Blunting Kit 100 rxns
    https://www.bioz.com/result/amplicons/product/New England Biolabs
    Average 92 stars, based on 3784 article reviews
    Price from $9.99 to $1999.99
    amplicons - by Bioz Stars, 2021-02
    92/100 stars

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    Images

    1) Product Images from "Mutation screening in 86 known X-linked mental retardation genes by droplet-based multiplex PCR and massive parallel sequencing"

    Article Title: Mutation screening in 86 known X-linked mental retardation genes by droplet-based multiplex PCR and massive parallel sequencing

    Journal: The HUGO Journal

    doi: 10.1007/s11568-010-9137-y

    The dependence of sequencing coverage of amplicons on their GC content ( a ) or amplicon length ( b ). The amplicons were grouped depending on their GC content ( a ) or length ( b ), the distribution of sequencing coverage within each group is shown as boxplot
    Figure Legend Snippet: The dependence of sequencing coverage of amplicons on their GC content ( a ) or amplicon length ( b ). The amplicons were grouped depending on their GC content ( a ) or length ( b ), the distribution of sequencing coverage within each group is shown as boxplot

    Techniques Used: Sequencing, Amplification

    Normalised sequencing coverage distribution. a Normalised coverage cumulative distribution of the sequenced bases within all amplicons. b Normalised coverage cumulative distribution of all amplicons. Normalised coverage is the absolute coverage divided by the mean coverage. Amplicon coverage is the median coverage of all the bases within the amplicon. Solid lines represent the average among all the 24 samples and the dashed lines represent 25th and 75th percentiles
    Figure Legend Snippet: Normalised sequencing coverage distribution. a Normalised coverage cumulative distribution of the sequenced bases within all amplicons. b Normalised coverage cumulative distribution of all amplicons. Normalised coverage is the absolute coverage divided by the mean coverage. Amplicon coverage is the median coverage of all the bases within the amplicon. Solid lines represent the average among all the 24 samples and the dashed lines represent 25th and 75th percentiles

    Techniques Used: Sequencing, Amplification

    2) Product Images from "Mutation screening in 86 known X-linked mental retardation genes by droplet-based multiplex PCR and massive parallel sequencing"

    Article Title: Mutation screening in 86 known X-linked mental retardation genes by droplet-based multiplex PCR and massive parallel sequencing

    Journal: The HUGO Journal

    doi: 10.1007/s11568-010-9137-y

    The dependence of sequencing coverage of amplicons on their GC content ( a ) or amplicon length ( b ). The amplicons were grouped depending on their GC content ( a ) or length ( b ), the distribution of sequencing coverage within each group is shown as boxplot
    Figure Legend Snippet: The dependence of sequencing coverage of amplicons on their GC content ( a ) or amplicon length ( b ). The amplicons were grouped depending on their GC content ( a ) or length ( b ), the distribution of sequencing coverage within each group is shown as boxplot

    Techniques Used: Sequencing, Amplification

    Normalised sequencing coverage distribution. a Normalised coverage cumulative distribution of the sequenced bases within all amplicons. b Normalised coverage cumulative distribution of all amplicons. Normalised coverage is the absolute coverage divided by the mean coverage. Amplicon coverage is the median coverage of all the bases within the amplicon. Solid lines represent the average among all the 24 samples and the dashed lines represent 25th and 75th percentiles
    Figure Legend Snippet: Normalised sequencing coverage distribution. a Normalised coverage cumulative distribution of the sequenced bases within all amplicons. b Normalised coverage cumulative distribution of all amplicons. Normalised coverage is the absolute coverage divided by the mean coverage. Amplicon coverage is the median coverage of all the bases within the amplicon. Solid lines represent the average among all the 24 samples and the dashed lines represent 25th and 75th percentiles

    Techniques Used: Sequencing, Amplification

    3) Product Images from "Identification of focally amplified lineage-specific super-enhancers in human epithelial cancers"

    Article Title: Identification of focally amplified lineage-specific super-enhancers in human epithelial cancers

    Journal: Nature genetics

    doi: 10.1038/ng.3470

    CRISPR/Cas9 mediated deletion of the e3 enhancer impairs the oncogenic effect of the e3 enhancer amplification (a) Upper: design of CRISPR/Cas9 mediated deletion of the e3 enhancer. Primers used to validate the e3 enhancer deletion are indicated. Bottom: Gel pictures of PCR amplification of genomic DNA using primers outside and inside the e3 enhancer region. sg-Empty: no sgRNA; sg-Control: a pair of sgRNAs that are predicted to not recognize any genomic regions; sg-e3del #1 and sg-e3del #2: two separate pairs of sgRNAs recognizing boundaries of the e3 enhancer region. PCR products were cloned into individual vectors and sequenced. Sequencing results represent the deletions induced by sg-e3del #1 (b) and sg-e3del #2 (c) . The expression level of MYC ± SEM as measured by quantitative PCR (n = 2) (d) , the cellular transformation efficiency ± SEM as measured by anchorage-independent growth (n = 3) (e) and the cellular proliferation rate ± SEM as measured by clonogenic assays (n = 3) (f) in NCI-H2009 cells with and without CRISPR/Cas9 mediated deletion of the e3 enhancer. The P -value is derived from a t-test; (*) p ≤0.05; (**) p ≤0.01. (g) Schematic representation of genomic structural variants activating MYC expression in cancer.
    Figure Legend Snippet: CRISPR/Cas9 mediated deletion of the e3 enhancer impairs the oncogenic effect of the e3 enhancer amplification (a) Upper: design of CRISPR/Cas9 mediated deletion of the e3 enhancer. Primers used to validate the e3 enhancer deletion are indicated. Bottom: Gel pictures of PCR amplification of genomic DNA using primers outside and inside the e3 enhancer region. sg-Empty: no sgRNA; sg-Control: a pair of sgRNAs that are predicted to not recognize any genomic regions; sg-e3del #1 and sg-e3del #2: two separate pairs of sgRNAs recognizing boundaries of the e3 enhancer region. PCR products were cloned into individual vectors and sequenced. Sequencing results represent the deletions induced by sg-e3del #1 (b) and sg-e3del #2 (c) . The expression level of MYC ± SEM as measured by quantitative PCR (n = 2) (d) , the cellular transformation efficiency ± SEM as measured by anchorage-independent growth (n = 3) (e) and the cellular proliferation rate ± SEM as measured by clonogenic assays (n = 3) (f) in NCI-H2009 cells with and without CRISPR/Cas9 mediated deletion of the e3 enhancer. The P -value is derived from a t-test; (*) p ≤0.05; (**) p ≤0.01. (g) Schematic representation of genomic structural variants activating MYC expression in cancer.

    Techniques Used: CRISPR, Amplification, Polymerase Chain Reaction, Clone Assay, Sequencing, Expressing, Real-time Polymerase Chain Reaction, Transformation Assay, Derivative Assay

    Identification of transcription factors required for the activity of the e3 enhancer (a) Enhancer activity ± SEM of small fragments (a-f) of the e3 enhancer as assessed by luciferase reporter assays (n = 3) in A549 LUAD cells. The fragments c and d show comparable enhancer activity relative to the intact e3 enhancer, while other fragments show minimal enhancer activity. The P -value is derived from a t-test; (***) p ≤0.001. (b) Transcription factor DNA recognition motifs are identified in the mini-e3 enhancer region that is defined by the c and d fragments overlap. (c) The luciferase reporter expression level ± SEM after deletion of individual transcription factor motif sequence in the e3 regions. The P -value is derived from a t-test (n = 3); (**) p ≤0.01; (***) p ≤0.001. (d) Luciferase reporter expression level ± SEM after silencing NFE2L2 or CEBPB by siRNA (n = 3). The P -value is derived from a t-test; (*) p ≤0.05; (**) p ≤0.01. (e) ChIP-seq profile of NFE2L2 and CEBPB in the e1–e5 enhancer regions in A549 cells.
    Figure Legend Snippet: Identification of transcription factors required for the activity of the e3 enhancer (a) Enhancer activity ± SEM of small fragments (a-f) of the e3 enhancer as assessed by luciferase reporter assays (n = 3) in A549 LUAD cells. The fragments c and d show comparable enhancer activity relative to the intact e3 enhancer, while other fragments show minimal enhancer activity. The P -value is derived from a t-test; (***) p ≤0.001. (b) Transcription factor DNA recognition motifs are identified in the mini-e3 enhancer region that is defined by the c and d fragments overlap. (c) The luciferase reporter expression level ± SEM after deletion of individual transcription factor motif sequence in the e3 regions. The P -value is derived from a t-test (n = 3); (**) p ≤0.01; (***) p ≤0.001. (d) Luciferase reporter expression level ± SEM after silencing NFE2L2 or CEBPB by siRNA (n = 3). The P -value is derived from a t-test; (*) p ≤0.05; (**) p ≤0.01. (e) ChIP-seq profile of NFE2L2 and CEBPB in the e1–e5 enhancer regions in A549 cells.

    Techniques Used: Activity Assay, Luciferase, Derivative Assay, Expressing, Sequencing, Chromatin Immunoprecipitation

    4) Product Images from "Identification of focally amplified lineage-specific super-enhancers in human epithelial cancers"

    Article Title: Identification of focally amplified lineage-specific super-enhancers in human epithelial cancers

    Journal: Nature genetics

    doi: 10.1038/ng.3470

    CRISPR/Cas9 mediated deletion of the e3 enhancer impairs the oncogenic effect of the e3 enhancer amplification (a) Upper: design of CRISPR/Cas9 mediated deletion of the e3 enhancer. Primers used to validate the e3 enhancer deletion are indicated. Bottom: Gel pictures of PCR amplification of genomic DNA using primers outside and inside the e3 enhancer region. sg-Empty: no sgRNA; sg-Control: a pair of sgRNAs that are predicted to not recognize any genomic regions; sg-e3del #1 and sg-e3del #2: two separate pairs of sgRNAs recognizing boundaries of the e3 enhancer region. PCR products were cloned into individual vectors and sequenced. Sequencing results represent the deletions induced by sg-e3del #1 (b) and sg-e3del #2 (c) . The expression level of MYC ± SEM as measured by quantitative PCR (n = 2) (d) , the cellular transformation efficiency ± SEM as measured by anchorage-independent growth (n = 3) (e) and the cellular proliferation rate ± SEM as measured by clonogenic assays (n = 3) (f) in NCI-H2009 cells with and without CRISPR/Cas9 mediated deletion of the e3 enhancer. The P -value is derived from a t-test; (*) p ≤0.05; (**) p ≤0.01. (g) Schematic representation of genomic structural variants activating MYC expression in cancer.
    Figure Legend Snippet: CRISPR/Cas9 mediated deletion of the e3 enhancer impairs the oncogenic effect of the e3 enhancer amplification (a) Upper: design of CRISPR/Cas9 mediated deletion of the e3 enhancer. Primers used to validate the e3 enhancer deletion are indicated. Bottom: Gel pictures of PCR amplification of genomic DNA using primers outside and inside the e3 enhancer region. sg-Empty: no sgRNA; sg-Control: a pair of sgRNAs that are predicted to not recognize any genomic regions; sg-e3del #1 and sg-e3del #2: two separate pairs of sgRNAs recognizing boundaries of the e3 enhancer region. PCR products were cloned into individual vectors and sequenced. Sequencing results represent the deletions induced by sg-e3del #1 (b) and sg-e3del #2 (c) . The expression level of MYC ± SEM as measured by quantitative PCR (n = 2) (d) , the cellular transformation efficiency ± SEM as measured by anchorage-independent growth (n = 3) (e) and the cellular proliferation rate ± SEM as measured by clonogenic assays (n = 3) (f) in NCI-H2009 cells with and without CRISPR/Cas9 mediated deletion of the e3 enhancer. The P -value is derived from a t-test; (*) p ≤0.05; (**) p ≤0.01. (g) Schematic representation of genomic structural variants activating MYC expression in cancer.

    Techniques Used: CRISPR, Amplification, Polymerase Chain Reaction, Clone Assay, Sequencing, Expressing, Real-time Polymerase Chain Reaction, Transformation Assay, Derivative Assay

    KRAB-dCas9 mediated repression of the e3 enhancer reveals MYC as a direct target (a) Upper: the design of KRAB-dCas9 mediated repression of the e3 enhancer. Bottom: ChIP-seq of H3K27ac in NCI-H2009 cells with and without KRAB-dCas9 enhancer repression. p300 ChIP-seq profile in parental NCI-H2009 cells indicates the e3 enhancer region. sg-Empty: no sgRNA; sg-Control: sgRNA that is predicted to not recognize any genomic regions; sg-e3KRAB #1 and sg-e3KRAB #2: two separate sgRNAs recognizing the e3 enhancer region. (b) The expression level of MYC ± SEM as measured by quantitative PCR in NCI-H2009 cells with and without KRAB-dCas9 mediated repression of the e3 enhancer (n = 2). (c) GSEA analysis of RNA-seq data generated in NIC-H2009 cells with and without KRAB-dCas9 mediated e3 enhancer repression reveals that genes regulated by e3 repression are enriched in MYC target genes identified by previous studies 44 – 47 . The cellular transformation efficiency ± SEM as measured by anchorage-independent growth (n = 3) (d) and the cellular proliferation rate ± SEM as measured by clonogenic assays (n = 3) (e) in NCI-H2009 cells with and without KRAB-dCas9 mediated repression of the e3 enhancer. The P -value is derived from a t-test; (*) p ≤0.05; (**) p ≤0.01; (***) p ≤0.001.
    Figure Legend Snippet: KRAB-dCas9 mediated repression of the e3 enhancer reveals MYC as a direct target (a) Upper: the design of KRAB-dCas9 mediated repression of the e3 enhancer. Bottom: ChIP-seq of H3K27ac in NCI-H2009 cells with and without KRAB-dCas9 enhancer repression. p300 ChIP-seq profile in parental NCI-H2009 cells indicates the e3 enhancer region. sg-Empty: no sgRNA; sg-Control: sgRNA that is predicted to not recognize any genomic regions; sg-e3KRAB #1 and sg-e3KRAB #2: two separate sgRNAs recognizing the e3 enhancer region. (b) The expression level of MYC ± SEM as measured by quantitative PCR in NCI-H2009 cells with and without KRAB-dCas9 mediated repression of the e3 enhancer (n = 2). (c) GSEA analysis of RNA-seq data generated in NIC-H2009 cells with and without KRAB-dCas9 mediated e3 enhancer repression reveals that genes regulated by e3 repression are enriched in MYC target genes identified by previous studies 44 – 47 . The cellular transformation efficiency ± SEM as measured by anchorage-independent growth (n = 3) (d) and the cellular proliferation rate ± SEM as measured by clonogenic assays (n = 3) (e) in NCI-H2009 cells with and without KRAB-dCas9 mediated repression of the e3 enhancer. The P -value is derived from a t-test; (*) p ≤0.05; (**) p ≤0.01; (***) p ≤0.001.

    Techniques Used: Chromatin Immunoprecipitation, Expressing, Real-time Polymerase Chain Reaction, RNA Sequencing Assay, Generated, Transformation Assay, Derivative Assay

    5) Product Images from "Identification of focally amplified lineage-specific super-enhancers in human epithelial cancers"

    Article Title: Identification of focally amplified lineage-specific super-enhancers in human epithelial cancers

    Journal: Nature genetics

    doi: 10.1038/ng.3470

    CRISPR/Cas9 mediated deletion of the e3 enhancer impairs the oncogenic effect of the e3 enhancer amplification (a) Upper: design of CRISPR/Cas9 mediated deletion of the e3 enhancer. Primers used to validate the e3 enhancer deletion are indicated. Bottom: Gel pictures of PCR amplification of genomic DNA using primers outside and inside the e3 enhancer region. sg-Empty: no sgRNA; sg-Control: a pair of sgRNAs that are predicted to not recognize any genomic regions; sg-e3del #1 and sg-e3del #2: two separate pairs of sgRNAs recognizing boundaries of the e3 enhancer region. PCR products were cloned into individual vectors and sequenced. Sequencing results represent the deletions induced by sg-e3del #1 (b) and sg-e3del #2 (c) . The expression level of MYC ± SEM as measured by quantitative PCR (n = 2) (d) , the cellular transformation efficiency ± SEM as measured by anchorage-independent growth (n = 3) (e) and the cellular proliferation rate ± SEM as measured by clonogenic assays (n = 3) (f) in NCI-H2009 cells with and without CRISPR/Cas9 mediated deletion of the e3 enhancer. The P -value is derived from a t-test; (*) p ≤0.05; (**) p ≤0.01. (g) Schematic representation of genomic structural variants activating MYC expression in cancer.
    Figure Legend Snippet: CRISPR/Cas9 mediated deletion of the e3 enhancer impairs the oncogenic effect of the e3 enhancer amplification (a) Upper: design of CRISPR/Cas9 mediated deletion of the e3 enhancer. Primers used to validate the e3 enhancer deletion are indicated. Bottom: Gel pictures of PCR amplification of genomic DNA using primers outside and inside the e3 enhancer region. sg-Empty: no sgRNA; sg-Control: a pair of sgRNAs that are predicted to not recognize any genomic regions; sg-e3del #1 and sg-e3del #2: two separate pairs of sgRNAs recognizing boundaries of the e3 enhancer region. PCR products were cloned into individual vectors and sequenced. Sequencing results represent the deletions induced by sg-e3del #1 (b) and sg-e3del #2 (c) . The expression level of MYC ± SEM as measured by quantitative PCR (n = 2) (d) , the cellular transformation efficiency ± SEM as measured by anchorage-independent growth (n = 3) (e) and the cellular proliferation rate ± SEM as measured by clonogenic assays (n = 3) (f) in NCI-H2009 cells with and without CRISPR/Cas9 mediated deletion of the e3 enhancer. The P -value is derived from a t-test; (*) p ≤0.05; (**) p ≤0.01. (g) Schematic representation of genomic structural variants activating MYC expression in cancer.

    Techniques Used: CRISPR, Amplification, Polymerase Chain Reaction, Clone Assay, Sequencing, Expressing, Real-time Polymerase Chain Reaction, Transformation Assay, Derivative Assay

    The activity of the MYC -LASE is predominantly driven by the e3 constituent enhancer (a) H3K27ac, p300 binding and DNase I hypersensitivity profiles in A549, NCI-H2009 and NCI-H358 cells reveal the constituent enhancers e1-e5 within the super-enhancer region. (b) Luciferase reporter assay (n = 3) measuring enhancer activity of e1-e5 in A549, NCI-H358 and NCI-H2009 lung adenocarcinoma cells. The pGL3 plasmid without the enhancer region (Empty) is used as a negative control. ( Y -axis) Relative Luciferase units are normalized to Renilla signal ± SEM. The P -value is derived from a t-test; (**) p ≤ p0.01; (***) p ≤0.001. (c) Enhancer activity of a duplicated e3 enhancer (2×e3) ± SEM as measured by luciferase reporter assay (n = 3). The P -value is derived from a t-test; (***) p ≤0.001.
    Figure Legend Snippet: The activity of the MYC -LASE is predominantly driven by the e3 constituent enhancer (a) H3K27ac, p300 binding and DNase I hypersensitivity profiles in A549, NCI-H2009 and NCI-H358 cells reveal the constituent enhancers e1-e5 within the super-enhancer region. (b) Luciferase reporter assay (n = 3) measuring enhancer activity of e1-e5 in A549, NCI-H358 and NCI-H2009 lung adenocarcinoma cells. The pGL3 plasmid without the enhancer region (Empty) is used as a negative control. ( Y -axis) Relative Luciferase units are normalized to Renilla signal ± SEM. The P -value is derived from a t-test; (**) p ≤ p0.01; (***) p ≤0.001. (c) Enhancer activity of a duplicated e3 enhancer (2×e3) ± SEM as measured by luciferase reporter assay (n = 3). The P -value is derived from a t-test; (***) p ≤0.001.

    Techniques Used: Activity Assay, Binding Assay, Luciferase, Reporter Assay, Plasmid Preparation, Negative Control, Derivative Assay

    Identification of transcription factors required for the activity of the e3 enhancer (a) Enhancer activity ± SEM of small fragments (a-f) of the e3 enhancer as assessed by luciferase reporter assays (n = 3) in A549 LUAD cells. The fragments c and d show comparable enhancer activity relative to the intact e3 enhancer, while other fragments show minimal enhancer activity. The P -value is derived from a t-test; (***) p ≤0.001. (b) Transcription factor DNA recognition motifs are identified in the mini-e3 enhancer region that is defined by the c and d fragments overlap. (c) The luciferase reporter expression level ± SEM after deletion of individual transcription factor motif sequence in the e3 regions. The P -value is derived from a t-test (n = 3); (**) p ≤0.01; (***) p ≤0.001. (d) Luciferase reporter expression level ± SEM after silencing NFE2L2 or CEBPB by siRNA (n = 3). The P -value is derived from a t-test; (*) p ≤0.05; (**) p ≤0.01. (e) ChIP-seq profile of NFE2L2 and CEBPB in the e1–e5 enhancer regions in A549 cells.
    Figure Legend Snippet: Identification of transcription factors required for the activity of the e3 enhancer (a) Enhancer activity ± SEM of small fragments (a-f) of the e3 enhancer as assessed by luciferase reporter assays (n = 3) in A549 LUAD cells. The fragments c and d show comparable enhancer activity relative to the intact e3 enhancer, while other fragments show minimal enhancer activity. The P -value is derived from a t-test; (***) p ≤0.001. (b) Transcription factor DNA recognition motifs are identified in the mini-e3 enhancer region that is defined by the c and d fragments overlap. (c) The luciferase reporter expression level ± SEM after deletion of individual transcription factor motif sequence in the e3 regions. The P -value is derived from a t-test (n = 3); (**) p ≤0.01; (***) p ≤0.001. (d) Luciferase reporter expression level ± SEM after silencing NFE2L2 or CEBPB by siRNA (n = 3). The P -value is derived from a t-test; (*) p ≤0.05; (**) p ≤0.01. (e) ChIP-seq profile of NFE2L2 and CEBPB in the e1–e5 enhancer regions in A549 cells.

    Techniques Used: Activity Assay, Luciferase, Derivative Assay, Expressing, Sequencing, Chromatin Immunoprecipitation

    KRAB-dCas9 mediated repression of the e3 enhancer reveals MYC as a direct target (a) Upper: the design of KRAB-dCas9 mediated repression of the e3 enhancer. Bottom: ChIP-seq of H3K27ac in NCI-H2009 cells with and without KRAB-dCas9 enhancer repression. p300 ChIP-seq profile in parental NCI-H2009 cells indicates the e3 enhancer region. sg-Empty: no sgRNA; sg-Control: sgRNA that is predicted to not recognize any genomic regions; sg-e3KRAB #1 and sg-e3KRAB #2: two separate sgRNAs recognizing the e3 enhancer region. (b) The expression level of MYC ± SEM as measured by quantitative PCR in NCI-H2009 cells with and without KRAB-dCas9 mediated repression of the e3 enhancer (n = 2). (c) GSEA analysis of RNA-seq data generated in NIC-H2009 cells with and without KRAB-dCas9 mediated e3 enhancer repression reveals that genes regulated by e3 repression are enriched in MYC target genes identified by previous studies 44 – 47 . The cellular transformation efficiency ± SEM as measured by anchorage-independent growth (n = 3) (d) and the cellular proliferation rate ± SEM as measured by clonogenic assays (n = 3) (e) in NCI-H2009 cells with and without KRAB-dCas9 mediated repression of the e3 enhancer. The P -value is derived from a t-test; (*) p ≤0.05; (**) p ≤0.01; (***) p ≤0.001.
    Figure Legend Snippet: KRAB-dCas9 mediated repression of the e3 enhancer reveals MYC as a direct target (a) Upper: the design of KRAB-dCas9 mediated repression of the e3 enhancer. Bottom: ChIP-seq of H3K27ac in NCI-H2009 cells with and without KRAB-dCas9 enhancer repression. p300 ChIP-seq profile in parental NCI-H2009 cells indicates the e3 enhancer region. sg-Empty: no sgRNA; sg-Control: sgRNA that is predicted to not recognize any genomic regions; sg-e3KRAB #1 and sg-e3KRAB #2: two separate sgRNAs recognizing the e3 enhancer region. (b) The expression level of MYC ± SEM as measured by quantitative PCR in NCI-H2009 cells with and without KRAB-dCas9 mediated repression of the e3 enhancer (n = 2). (c) GSEA analysis of RNA-seq data generated in NIC-H2009 cells with and without KRAB-dCas9 mediated e3 enhancer repression reveals that genes regulated by e3 repression are enriched in MYC target genes identified by previous studies 44 – 47 . The cellular transformation efficiency ± SEM as measured by anchorage-independent growth (n = 3) (d) and the cellular proliferation rate ± SEM as measured by clonogenic assays (n = 3) (e) in NCI-H2009 cells with and without KRAB-dCas9 mediated repression of the e3 enhancer. The P -value is derived from a t-test; (*) p ≤0.05; (**) p ≤0.01; (***) p ≤0.001.

    Techniques Used: Chromatin Immunoprecipitation, Expressing, Real-time Polymerase Chain Reaction, RNA Sequencing Assay, Generated, Transformation Assay, Derivative Assay

    6) Product Images from "ERCC1-XPF Interacts with Topoisomerase IIβ to Facilitate the Repair of Activity-induced DNA Breaks"

    Article Title: ERCC1-XPF Interacts with Topoisomerase IIβ to Facilitate the Repair of Activity-induced DNA Breaks

    Journal: bioRxiv

    doi: 10.1101/2020.01.03.892703

    Genome-wide mapping of DSBs in tRA-treated and Ercc1 −/− MEFs. (A) . Genome-wide enrichment (read count; R.C.) of DNA DSBs (normalized per million mapped reads) on -/+2Kb flanking the TSS in untreated (Untr.), (B) . tRA-treated (tRA) and (C) . Ercc1 −/− MEFs. (D) . BLESS signals quantified by qPCR on the tRA-inducible Cfh , Rarb and Hs3st1 gene promoters, the tRA non-inducible Chordc1 gene promoter, as well as on a non-transcribed intergenic region (-) region in untreated MEFs (Untr.) or MEFs treated with tRA (tRA) or tRA and merbarone (tRA/merb). (E) . Enrichment of ERCC1, TOP IIβ and CTCF proteins on DSB-containing genomic fragments derived from primary MEFs. (F) . Venn’s logic diagrams representing the number of transcription-associated DNA DSBs on XPF-bound genomic sites in untreated (Untr.) and tRA-treated MEFs. (G) . Probability of XPF recruitment on genes by means of log^2RNA*log^2Breaks variable from the RNA-Seq and BLISS data. ( H ). BLESS signals quantified by qPCR on Rarb2 gene promoter in tRA-treated wt. and Ercc1 −/− MEFs. ( I ). BLESS signals quantified by qPCR on the promoters of PrlR and Dio1 genes known to be expressed in the P15 Ercc1 −/− and wt. livers and on the promoter of non-expressed Neun gene. ( J ). BLESS signals quantified by qPCR on the promoter of Dhfr and Prnp genes known to be expressed in the P15 Ercc1 −/− and wt. cerebella and on the promoter of the non-expressed Alb gene. Error bars indicate S.E.M. among n > three biological replicates. Asterisk indicates the significance set at p-value: *≤0.05, **≤0.01, ***≤0.001 (two-tailed Student’s t-test).
    Figure Legend Snippet: Genome-wide mapping of DSBs in tRA-treated and Ercc1 −/− MEFs. (A) . Genome-wide enrichment (read count; R.C.) of DNA DSBs (normalized per million mapped reads) on -/+2Kb flanking the TSS in untreated (Untr.), (B) . tRA-treated (tRA) and (C) . Ercc1 −/− MEFs. (D) . BLESS signals quantified by qPCR on the tRA-inducible Cfh , Rarb and Hs3st1 gene promoters, the tRA non-inducible Chordc1 gene promoter, as well as on a non-transcribed intergenic region (-) region in untreated MEFs (Untr.) or MEFs treated with tRA (tRA) or tRA and merbarone (tRA/merb). (E) . Enrichment of ERCC1, TOP IIβ and CTCF proteins on DSB-containing genomic fragments derived from primary MEFs. (F) . Venn’s logic diagrams representing the number of transcription-associated DNA DSBs on XPF-bound genomic sites in untreated (Untr.) and tRA-treated MEFs. (G) . Probability of XPF recruitment on genes by means of log^2RNA*log^2Breaks variable from the RNA-Seq and BLISS data. ( H ). BLESS signals quantified by qPCR on Rarb2 gene promoter in tRA-treated wt. and Ercc1 −/− MEFs. ( I ). BLESS signals quantified by qPCR on the promoters of PrlR and Dio1 genes known to be expressed in the P15 Ercc1 −/− and wt. livers and on the promoter of non-expressed Neun gene. ( J ). BLESS signals quantified by qPCR on the promoter of Dhfr and Prnp genes known to be expressed in the P15 Ercc1 −/− and wt. cerebella and on the promoter of the non-expressed Alb gene. Error bars indicate S.E.M. among n > three biological replicates. Asterisk indicates the significance set at p-value: *≤0.05, **≤0.01, ***≤0.001 (two-tailed Student’s t-test).

    Techniques Used: Genome Wide, Real-time Polymerase Chain Reaction, Derivative Assay, RNA Sequencing Assay, Two Tailed Test

    7) Product Images from "Identification of focally amplified lineage-specific super-enhancers in human epithelial cancers"

    Article Title: Identification of focally amplified lineage-specific super-enhancers in human epithelial cancers

    Journal: Nature genetics

    doi: 10.1038/ng.3470

    CRISPR/Cas9 mediated deletion of the e3 enhancer impairs the oncogenic effect of the e3 enhancer amplification (a) Upper: design of CRISPR/Cas9 mediated deletion of the e3 enhancer. Primers used to validate the e3 enhancer deletion are indicated. Bottom: Gel pictures of PCR amplification of genomic DNA using primers outside and inside the e3 enhancer region. sg-Empty: no sgRNA; sg-Control: a pair of sgRNAs that are predicted to not recognize any genomic regions; sg-e3del #1 and sg-e3del #2: two separate pairs of sgRNAs recognizing boundaries of the e3 enhancer region. PCR products were cloned into individual vectors and sequenced. Sequencing results represent the deletions induced by sg-e3del #1 (b) and sg-e3del #2 (c) . The expression level of MYC ± SEM as measured by quantitative PCR (n = 2) (d) , the cellular transformation efficiency ± SEM as measured by anchorage-independent growth (n = 3) (e) and the cellular proliferation rate ± SEM as measured by clonogenic assays (n = 3) (f) in NCI-H2009 cells with and without CRISPR/Cas9 mediated deletion of the e3 enhancer. The P -value is derived from a t-test; (*) p ≤0.05; (**) p ≤0.01. (g) Schematic representation of genomic structural variants activating MYC expression in cancer.
    Figure Legend Snippet: CRISPR/Cas9 mediated deletion of the e3 enhancer impairs the oncogenic effect of the e3 enhancer amplification (a) Upper: design of CRISPR/Cas9 mediated deletion of the e3 enhancer. Primers used to validate the e3 enhancer deletion are indicated. Bottom: Gel pictures of PCR amplification of genomic DNA using primers outside and inside the e3 enhancer region. sg-Empty: no sgRNA; sg-Control: a pair of sgRNAs that are predicted to not recognize any genomic regions; sg-e3del #1 and sg-e3del #2: two separate pairs of sgRNAs recognizing boundaries of the e3 enhancer region. PCR products were cloned into individual vectors and sequenced. Sequencing results represent the deletions induced by sg-e3del #1 (b) and sg-e3del #2 (c) . The expression level of MYC ± SEM as measured by quantitative PCR (n = 2) (d) , the cellular transformation efficiency ± SEM as measured by anchorage-independent growth (n = 3) (e) and the cellular proliferation rate ± SEM as measured by clonogenic assays (n = 3) (f) in NCI-H2009 cells with and without CRISPR/Cas9 mediated deletion of the e3 enhancer. The P -value is derived from a t-test; (*) p ≤0.05; (**) p ≤0.01. (g) Schematic representation of genomic structural variants activating MYC expression in cancer.

    Techniques Used: CRISPR, Amplification, Polymerase Chain Reaction, Clone Assay, Sequencing, Expressing, Real-time Polymerase Chain Reaction, Transformation Assay, Derivative Assay

    KRAB-dCas9 mediated repression of the e3 enhancer reveals MYC as a direct target (a) Upper: the design of KRAB-dCas9 mediated repression of the e3 enhancer. Bottom: ChIP-seq of H3K27ac in NCI-H2009 cells with and without KRAB-dCas9 enhancer repression. p300 ChIP-seq profile in parental NCI-H2009 cells indicates the e3 enhancer region. sg-Empty: no sgRNA; sg-Control: sgRNA that is predicted to not recognize any genomic regions; sg-e3KRAB #1 and sg-e3KRAB #2: two separate sgRNAs recognizing the e3 enhancer region. (b) The expression level of MYC ± SEM as measured by quantitative PCR in NCI-H2009 cells with and without KRAB-dCas9 mediated repression of the e3 enhancer (n = 2). (c) GSEA analysis of RNA-seq data generated in NIC-H2009 cells with and without KRAB-dCas9 mediated e3 enhancer repression reveals that genes regulated by e3 repression are enriched in MYC target genes identified by previous studies 44 – 47 . The cellular transformation efficiency ± SEM as measured by anchorage-independent growth (n = 3) (d) and the cellular proliferation rate ± SEM as measured by clonogenic assays (n = 3) (e) in NCI-H2009 cells with and without KRAB-dCas9 mediated repression of the e3 enhancer. The P -value is derived from a t-test; (*) p ≤0.05; (**) p ≤0.01; (***) p ≤0.001.
    Figure Legend Snippet: KRAB-dCas9 mediated repression of the e3 enhancer reveals MYC as a direct target (a) Upper: the design of KRAB-dCas9 mediated repression of the e3 enhancer. Bottom: ChIP-seq of H3K27ac in NCI-H2009 cells with and without KRAB-dCas9 enhancer repression. p300 ChIP-seq profile in parental NCI-H2009 cells indicates the e3 enhancer region. sg-Empty: no sgRNA; sg-Control: sgRNA that is predicted to not recognize any genomic regions; sg-e3KRAB #1 and sg-e3KRAB #2: two separate sgRNAs recognizing the e3 enhancer region. (b) The expression level of MYC ± SEM as measured by quantitative PCR in NCI-H2009 cells with and without KRAB-dCas9 mediated repression of the e3 enhancer (n = 2). (c) GSEA analysis of RNA-seq data generated in NIC-H2009 cells with and without KRAB-dCas9 mediated e3 enhancer repression reveals that genes regulated by e3 repression are enriched in MYC target genes identified by previous studies 44 – 47 . The cellular transformation efficiency ± SEM as measured by anchorage-independent growth (n = 3) (d) and the cellular proliferation rate ± SEM as measured by clonogenic assays (n = 3) (e) in NCI-H2009 cells with and without KRAB-dCas9 mediated repression of the e3 enhancer. The P -value is derived from a t-test; (*) p ≤0.05; (**) p ≤0.01; (***) p ≤0.001.

    Techniques Used: Chromatin Immunoprecipitation, Expressing, Real-time Polymerase Chain Reaction, RNA Sequencing Assay, Generated, Transformation Assay, Derivative Assay

    8) Product Images from "An early cell shape transition drives evolutionary expansion of the human forebrain"

    Article Title: An early cell shape transition drives evolutionary expansion of the human forebrain

    Journal: bioRxiv

    doi: 10.1101/2020.07.04.188078

    Early forced expression of ZEB2 in human NE cells. A. Plasmid maps of the CRISPR homology-directed repair (HDR) templates used to target the AAVS1 safe-harbour locus in H9 hESC cells – top is the CAG-lox-STOP-lox-ZEB2-GFP-Flag inducible expression construct and bottom is the construct encoding CRE recombinase under the control of a tetracycline responsive promoter and the reverse tetracycline transactivator (rtTA) driven by the CAG promoter. B . UCSC Genome Browser view of the AAVS1 locus and CRISPR-Cas9 targeting strategy of intron 1 of PPP1R12C . The promoter-less splice-acceptor (SA), T2A peptide-linked “gene trap” is such that expression of the promoter-less selection cassette is driven by the endogenous PPP1R12C gene, thus effectively eliminating false-positive background arising from random integration. The panel reports the PCR genotyping strategy – upon successful targeting of the AAVS1 locus, while amplicon 1 is lost due to the size increase following insert integration, amplicons 2 and 3 are gained. The PCR gel shows successful genotyping of the colony used for all experiments shown. The asterisks mark unspecific bands C . Transgene induction test in H9 iZEB2 cells treated with and without doxycycline for 6 days and assayed by western blot for ZEB2, GFP and β-actin. D . Immunofluorescence images of 6-day induced and uninduced H9 iZEB2 cells stained for ZEB2 and DAPI, showing that doxycyline induction results in ZEB2 expression and nuclear translocation, without adverse effects on its localisation due to tagging with GFP. Scale bar: 20 µm E . Immunofluorescence images of 6-day induced and uninduced H9 iZEB2 cells stained for GFP, Vimentin and EpCAM. The data reveal a reduction in EpCAM expression and an increase in Vimentin expression following expression of ZEB2-GFP. Scale bar: 50 µm F . Immunofluorescence images of 6-day induced and uninduced H9 iZEB2 cells stained for DAPI, CDH1 and CDH2. The data reveal a reduction in CDH1 expression and an increase in CDH2 expression following induction. Scale bar: 50 µm. G . Brightfield images of induced (+ Dox) and uninduced (-Dox) H9 iZEB2 organoids and gorilla (G1) organoids at day 3 and day 5, showing indistinguishable tissue architecture between organoids at day 3, while day 5 organoids show neural buds that are more rounded in gorilla and ZEB2 induced vs uninduced organoids. Scale bar: 200 μm.
    Figure Legend Snippet: Early forced expression of ZEB2 in human NE cells. A. Plasmid maps of the CRISPR homology-directed repair (HDR) templates used to target the AAVS1 safe-harbour locus in H9 hESC cells – top is the CAG-lox-STOP-lox-ZEB2-GFP-Flag inducible expression construct and bottom is the construct encoding CRE recombinase under the control of a tetracycline responsive promoter and the reverse tetracycline transactivator (rtTA) driven by the CAG promoter. B . UCSC Genome Browser view of the AAVS1 locus and CRISPR-Cas9 targeting strategy of intron 1 of PPP1R12C . The promoter-less splice-acceptor (SA), T2A peptide-linked “gene trap” is such that expression of the promoter-less selection cassette is driven by the endogenous PPP1R12C gene, thus effectively eliminating false-positive background arising from random integration. The panel reports the PCR genotyping strategy – upon successful targeting of the AAVS1 locus, while amplicon 1 is lost due to the size increase following insert integration, amplicons 2 and 3 are gained. The PCR gel shows successful genotyping of the colony used for all experiments shown. The asterisks mark unspecific bands C . Transgene induction test in H9 iZEB2 cells treated with and without doxycycline for 6 days and assayed by western blot for ZEB2, GFP and β-actin. D . Immunofluorescence images of 6-day induced and uninduced H9 iZEB2 cells stained for ZEB2 and DAPI, showing that doxycyline induction results in ZEB2 expression and nuclear translocation, without adverse effects on its localisation due to tagging with GFP. Scale bar: 20 µm E . Immunofluorescence images of 6-day induced and uninduced H9 iZEB2 cells stained for GFP, Vimentin and EpCAM. The data reveal a reduction in EpCAM expression and an increase in Vimentin expression following expression of ZEB2-GFP. Scale bar: 50 µm F . Immunofluorescence images of 6-day induced and uninduced H9 iZEB2 cells stained for DAPI, CDH1 and CDH2. The data reveal a reduction in CDH1 expression and an increase in CDH2 expression following induction. Scale bar: 50 µm. G . Brightfield images of induced (+ Dox) and uninduced (-Dox) H9 iZEB2 organoids and gorilla (G1) organoids at day 3 and day 5, showing indistinguishable tissue architecture between organoids at day 3, while day 5 organoids show neural buds that are more rounded in gorilla and ZEB2 induced vs uninduced organoids. Scale bar: 200 μm.

    Techniques Used: Expressing, Plasmid Preparation, CRISPR, Construct, Selection, Polymerase Chain Reaction, Amplification, Western Blot, Immunofluorescence, Staining, Translocation Assay

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