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    Covaris 200 bp fragments
    Direct activation of zygotic transcription by Pou5f3 and Sox19b is non-additive. a. left: live phenotypes of MZ Pou5f3 (p), Sox19b (s), double mutant (d), and the wild-type (w). Right – RNA-seq experiments; samples collected in one experiment have the same color. Scale bar <t>200</t> μm. b. Expression of the top genes regulated by enhancers types 1-4 in the WT, single and double mutants (compare to Fig.4d-g , which shows H3K27ac in the regulatory regions of the same genes). Note, that type 4 gene noggin1 is strongly upregulated in the double mutant. c. RNA-sense plots for zygotic genes downregulated in the indicated mutants. Y-axis – time profile groups by WT switch up time, X-axis – groups of downregulated transcripts per time point. FET-Fisher’s Exact Test. d. The scope of ZGA delays in single and double mutants, per switch UP time group. Zygotic transcripts were scored as repressed, if they were downregulated in at least one point at or after the switch up (to the right from the white line in Fig. 5c ). Numbers (n) of repressed transcripts (n total transcripts) per group are shown above the diagrams. Group of transcripts repressed in both single mutants, but not in double mutant (s,p
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    Images

    1) Product Images from "Pluripotency factors select gene expression repertoire at Zygotic Genome Activation"

    Article Title: Pluripotency factors select gene expression repertoire at Zygotic Genome Activation

    Journal: bioRxiv

    doi: 10.1101/2020.02.16.949362

    Direct activation of zygotic transcription by Pou5f3 and Sox19b is non-additive. a. left: live phenotypes of MZ Pou5f3 (p), Sox19b (s), double mutant (d), and the wild-type (w). Right – RNA-seq experiments; samples collected in one experiment have the same color. Scale bar 200 μm. b. Expression of the top genes regulated by enhancers types 1-4 in the WT, single and double mutants (compare to Fig.4d-g , which shows H3K27ac in the regulatory regions of the same genes). Note, that type 4 gene noggin1 is strongly upregulated in the double mutant. c. RNA-sense plots for zygotic genes downregulated in the indicated mutants. Y-axis – time profile groups by WT switch up time, X-axis – groups of downregulated transcripts per time point. FET-Fisher’s Exact Test. d. The scope of ZGA delays in single and double mutants, per switch UP time group. Zygotic transcripts were scored as repressed, if they were downregulated in at least one point at or after the switch up (to the right from the white line in Fig. 5c ). Numbers (n) of repressed transcripts (n total transcripts) per group are shown above the diagrams. Group of transcripts repressed in both single mutants, but not in double mutant (s,p
    Figure Legend Snippet: Direct activation of zygotic transcription by Pou5f3 and Sox19b is non-additive. a. left: live phenotypes of MZ Pou5f3 (p), Sox19b (s), double mutant (d), and the wild-type (w). Right – RNA-seq experiments; samples collected in one experiment have the same color. Scale bar 200 μm. b. Expression of the top genes regulated by enhancers types 1-4 in the WT, single and double mutants (compare to Fig.4d-g , which shows H3K27ac in the regulatory regions of the same genes). Note, that type 4 gene noggin1 is strongly upregulated in the double mutant. c. RNA-sense plots for zygotic genes downregulated in the indicated mutants. Y-axis – time profile groups by WT switch up time, X-axis – groups of downregulated transcripts per time point. FET-Fisher’s Exact Test. d. The scope of ZGA delays in single and double mutants, per switch UP time group. Zygotic transcripts were scored as repressed, if they were downregulated in at least one point at or after the switch up (to the right from the white line in Fig. 5c ). Numbers (n) of repressed transcripts (n total transcripts) per group are shown above the diagrams. Group of transcripts repressed in both single mutants, but not in double mutant (s,p

    Techniques Used: Activation Assay, Mutagenesis, RNA Sequencing Assay, Expressing

    Mutation in maternal sox19b gene causes developmental delay starting at ZGA. a . Disruption of the sox19b gene on chromosome 7 by introducing an 8 bp deletion with TALEN technique. We chose an 8 bp deletion after the codon for amino acid 11 which resulted in introduction of a premature stop codon after another 62 amino acids. b . Difference in cell size and nuclei number between WT and MZsox19b detectable at 3.0 hpf. SYTOX green-nuclei, rhodamine-phalloidin-submembrane cytoskeleton. To compare the duration of 10th cell cycle, we fixed WT and MZsox19b embryos every 15 min starting from 2.5 hpf till 4 hpf, and compared the size of the cells and number of nuclei between the genotypes. We could detect that the MZsox19b were still at the 512 cell stage (10th cell cycle), while the WT embryos proceeded to the 1024 cell stage. MZsox19b completed 10 th cell cycle within the next 15 minutes. c . Phenotypes of the WT and MZsox19b embryos at the indicated time points. Morphogenetic movements in zebrafish blastula start from doming of the yolk (white arrow) followed by epiboly. Double white arrows show the distance from epiboly border (white dotted line) to the vegetal pole. Involution of mesendodermal layer marks gastrulation onset (hollow arrowheads in the wild-type, 5.7 hpf and 6 hpf), is followed by shield formation (black arrow in the wild-type, 6 hpf). Gastrulation ends with tail bud formation (black arrow at 10 hpf, WT, and at 12 hpf in MZ sox19b ). Scale bar 200 μm. d . Altered developmental timing in MZ sox19b : 10 th cell cycle is delayed at
    Figure Legend Snippet: Mutation in maternal sox19b gene causes developmental delay starting at ZGA. a . Disruption of the sox19b gene on chromosome 7 by introducing an 8 bp deletion with TALEN technique. We chose an 8 bp deletion after the codon for amino acid 11 which resulted in introduction of a premature stop codon after another 62 amino acids. b . Difference in cell size and nuclei number between WT and MZsox19b detectable at 3.0 hpf. SYTOX green-nuclei, rhodamine-phalloidin-submembrane cytoskeleton. To compare the duration of 10th cell cycle, we fixed WT and MZsox19b embryos every 15 min starting from 2.5 hpf till 4 hpf, and compared the size of the cells and number of nuclei between the genotypes. We could detect that the MZsox19b were still at the 512 cell stage (10th cell cycle), while the WT embryos proceeded to the 1024 cell stage. MZsox19b completed 10 th cell cycle within the next 15 minutes. c . Phenotypes of the WT and MZsox19b embryos at the indicated time points. Morphogenetic movements in zebrafish blastula start from doming of the yolk (white arrow) followed by epiboly. Double white arrows show the distance from epiboly border (white dotted line) to the vegetal pole. Involution of mesendodermal layer marks gastrulation onset (hollow arrowheads in the wild-type, 5.7 hpf and 6 hpf), is followed by shield formation (black arrow in the wild-type, 6 hpf). Gastrulation ends with tail bud formation (black arrow at 10 hpf, WT, and at 12 hpf in MZ sox19b ). Scale bar 200 μm. d . Altered developmental timing in MZ sox19b : 10 th cell cycle is delayed at

    Techniques Used: Mutagenesis

    2) Product Images from "Tcf1 and Lef1 transcription factors establish CD8+ T cell identity through intrinsic HDAC activity"

    Article Title: Tcf1 and Lef1 transcription factors establish CD8+ T cell identity through intrinsic HDAC activity

    Journal: Nature immunology

    doi: 10.1038/ni.3456

    The Tcf1 HDAC activity is essential for establis hing CD8 +  T cell identity ( a ) Analysis of frequency (left) and numbers (right) of donor-derived CD45.2 + GFP + TCRβ + CD4 +  T cells in the spleens of BM chimeras that were reconstituted with  Tcf7 −/− Lef1 −/−  lineage-negative bond marrow cells retrovirally infected with empty vector (EV), WT p45 Tcf1, or Tcf1 Mut5aa retrovirus. ( b ) Quantitative RT-PCR analysis of gene expression (relative to  Hprt ) in wild-type (WT) splenic CD4 +  or CD8 +  T cells, splenic CD45.2 + GFP + TCRβ + CD8 +  T cells sorted from the EV-, WT p45 Tcf1-, or Tcf1 Mut5aa-complemented  Tcf7 −/− Lef1 −/−  BM chimeras. For each gene, its expression in EV-complemented cells was set as 1, and that in other cell types was normalized accordingly. ( c ) Immunoblot analysis of H3K27Ac and total H3 histone in histone protein extracted from splenic CD45.2 + GFP + TCRβ +  CD8 +  T cells sorted from the EV-, WT p45 Tcf1-, or Tcf1 Mut5aa-complemented  Tcf7 −/− Lef1 −/−  BM chimeras. ( d ) ChIP-qPCR analysis of relative H3K27Ac signals at select gene loci in splenic CD45.2 + GFP + TCRβ + CD8 +  T cells sorted from EV-, WT p45 Tcf1-, or Tcf1 Mut5aa-complemented  Tcf7 −/− Lef1 −/−  BM chimeras. For each gene locus, the relative H3K27Ac signal in EV-complemented cells was set as 1, and that in other cell types was normalized accordingly. Data are from 3 experiments ( a, b , means ± s.d., n = 4–5), or representative from 3 experiments ( c ), or from 2 experiments with each sample measured in duplicates ( d , means ± s.d.). #, p = 0.11; ##, p = 0.06; *, p
    Figure Legend Snippet: The Tcf1 HDAC activity is essential for establis hing CD8 + T cell identity ( a ) Analysis of frequency (left) and numbers (right) of donor-derived CD45.2 + GFP + TCRβ + CD4 + T cells in the spleens of BM chimeras that were reconstituted with Tcf7 −/− Lef1 −/− lineage-negative bond marrow cells retrovirally infected with empty vector (EV), WT p45 Tcf1, or Tcf1 Mut5aa retrovirus. ( b ) Quantitative RT-PCR analysis of gene expression (relative to Hprt ) in wild-type (WT) splenic CD4 + or CD8 + T cells, splenic CD45.2 + GFP + TCRβ + CD8 + T cells sorted from the EV-, WT p45 Tcf1-, or Tcf1 Mut5aa-complemented Tcf7 −/− Lef1 −/− BM chimeras. For each gene, its expression in EV-complemented cells was set as 1, and that in other cell types was normalized accordingly. ( c ) Immunoblot analysis of H3K27Ac and total H3 histone in histone protein extracted from splenic CD45.2 + GFP + TCRβ + CD8 + T cells sorted from the EV-, WT p45 Tcf1-, or Tcf1 Mut5aa-complemented Tcf7 −/− Lef1 −/− BM chimeras. ( d ) ChIP-qPCR analysis of relative H3K27Ac signals at select gene loci in splenic CD45.2 + GFP + TCRβ + CD8 + T cells sorted from EV-, WT p45 Tcf1-, or Tcf1 Mut5aa-complemented Tcf7 −/− Lef1 −/− BM chimeras. For each gene locus, the relative H3K27Ac signal in EV-complemented cells was set as 1, and that in other cell types was normalized accordingly. Data are from 3 experiments ( a, b , means ± s.d., n = 4–5), or representative from 3 experiments ( c ), or from 2 experiments with each sample measured in duplicates ( d , means ± s.d.). #, p = 0.11; ##, p = 0.06; *, p

    Techniques Used: Activity Assay, Derivative Assay, Infection, Plasmid Preparation, Quantitative RT-PCR, Expressing, Chromatin Immunoprecipitation, Real-time Polymerase Chain Reaction

    Tcf7 −/− Lef1 −/−  CD8 +  T cells exhibit histone hyperacetylation ( a ) ChIP-Seq analysis of H3K27Ac and H3K9Ac histone marks in CD4 +  or CD8 +  mature thymocytes sorted from wild-type (WT) mice or CD8 +  mature thymocytes sorted from  Tcf7 −/− Lef1 −/−  mice, with H3K27Ac and H3K9Ac profiles (normalize read counts) shown at the “−5 kb to +5 kb” regions flanking the TSSs of 108 genes in the CD4 +  T cell gene set. ( b ) H3K27Ac ChIP-Seq tracks at the  Cd4  and  Itgb3  gene loci in WT CD4 + , WT or  Tcf7 −/− Lef1 −/−  CD8 +  mature thymocytes (marked on top of the panels are the gene structures and transcriptional orientations), along with Tcf1 ChIP-Seq tracks in splenic CD8 +  T cells, where MACS-called Tcf1 binding peaks are marked by green rectangles. ( c ) H3K27Ac and H3K9Ac profiles (normalize read counts) at the “−5 kb to +5 kb” regions flanking the TSSs of 472 upregulated genes in  Tcf7 −/− Lef1 −/−  CD8 +  mature thymocytes. ( d ) H3K27Ac ChIP-seq tracks at the  Prdm1  and  Fasl  gene loci in WT and  Tcf7 −/− Lef1 −/−  CD8 +  mature thymocytes, along with Tcf1 ChIP-Seq tracks in splenic CD8 +  T cells. ( e ) ChIP-qPCR analysis of relative H3K27Ac signals (ChIP with H3K27Ac antibody normalized to ChIP with IgG) at the indicated genomic locations in splenic WT CD4 + , WT or  Tcf7 −/− Lef1 −/−  CD8 +  T cells. Data are from one experiment ( a-d ), or from 2 experiments with each sample measured in duplicate ( e , means ± s.d.).
    Figure Legend Snippet: Tcf7 −/− Lef1 −/− CD8 + T cells exhibit histone hyperacetylation ( a ) ChIP-Seq analysis of H3K27Ac and H3K9Ac histone marks in CD4 + or CD8 + mature thymocytes sorted from wild-type (WT) mice or CD8 + mature thymocytes sorted from Tcf7 −/− Lef1 −/− mice, with H3K27Ac and H3K9Ac profiles (normalize read counts) shown at the “−5 kb to +5 kb” regions flanking the TSSs of 108 genes in the CD4 + T cell gene set. ( b ) H3K27Ac ChIP-Seq tracks at the Cd4 and Itgb3 gene loci in WT CD4 + , WT or Tcf7 −/− Lef1 −/− CD8 + mature thymocytes (marked on top of the panels are the gene structures and transcriptional orientations), along with Tcf1 ChIP-Seq tracks in splenic CD8 + T cells, where MACS-called Tcf1 binding peaks are marked by green rectangles. ( c ) H3K27Ac and H3K9Ac profiles (normalize read counts) at the “−5 kb to +5 kb” regions flanking the TSSs of 472 upregulated genes in Tcf7 −/− Lef1 −/− CD8 + mature thymocytes. ( d ) H3K27Ac ChIP-seq tracks at the Prdm1 and Fasl gene loci in WT and Tcf7 −/− Lef1 −/− CD8 + mature thymocytes, along with Tcf1 ChIP-Seq tracks in splenic CD8 + T cells. ( e ) ChIP-qPCR analysis of relative H3K27Ac signals (ChIP with H3K27Ac antibody normalized to ChIP with IgG) at the indicated genomic locations in splenic WT CD4 + , WT or Tcf7 −/− Lef1 −/− CD8 + T cells. Data are from one experiment ( a-d ), or from 2 experiments with each sample measured in duplicate ( e , means ± s.d.).

    Techniques Used: Chromatin Immunoprecipitation, Mouse Assay, Magnetic Cell Separation, Binding Assay, Real-time Polymerase Chain Reaction

    Tcf1 is connected with histone acetylation status in CD8 +  T cells ( a ) Immunoblot analysis of total or modified H3 histones in histone protein extracted from splenic CD8 +  T cells sorted from  Tcf7 −/− Lef1 −/−  or control littermates. ( b ) H3K27Ac and H3K9Ac profiles (normalize read counts) of wild-type (WT) and  Tcf7 −/− Lef1 −/−  CD8 +  mature thymocytes within and outside the 7,807 high-confidence Tcf1 binding sites identified by Tcf1 ChIP-Seq analysis in splenic CD8 +  T cells. Data are representative of two experiments ( a ), or from one experiment ( b ).
    Figure Legend Snippet: Tcf1 is connected with histone acetylation status in CD8 + T cells ( a ) Immunoblot analysis of total or modified H3 histones in histone protein extracted from splenic CD8 + T cells sorted from Tcf7 −/− Lef1 −/− or control littermates. ( b ) H3K27Ac and H3K9Ac profiles (normalize read counts) of wild-type (WT) and Tcf7 −/− Lef1 −/− CD8 + mature thymocytes within and outside the 7,807 high-confidence Tcf1 binding sites identified by Tcf1 ChIP-Seq analysis in splenic CD8 + T cells. Data are representative of two experiments ( a ), or from one experiment ( b ).

    Techniques Used: Modification, Binding Assay, Chromatin Immunoprecipitation

    Tcf1 and Lef1 deficiency perturbs CD8 +  T cell integrity (a)  RNA-Seq analysis of genes upregulated (red) or downregulated (blue) in TCRβ hi CD24 − CD69 − CD8 +  mature thymocytes sorted from  Tcf7 −/− Tcf7 −/− Lef1 −/− , and control littermates, with the average FPKM values of two replicates of control (Ctrl)  vs. Tcf7 −/− Lef1 −/−  CD8 +  T cells shown in a scatterplot, where the green lines denote gene expression changes of ≥2 fold.  (b)  Select upregulated genes (right margin) in  Tcf7 −/− Lef1 −/− CD8 +  mature thymocytes relative to control and  Tcf7 −/−  CD8 +  thymocytes as shown in a heatmap.  (c)  GSEA showing enriched expression of genes in the CD4 +  T cell gene set in  Tcf7 −/− Lef1 −/−  CD8 +  mature thymocytes, with the enriched genes (red rectangle in the enrichment plot) displayed in a heatmap, where CD4 +  signature genes are highlighted in green.  (d)  Quantitative RT-PCR analysis of  Cd40lg, St8sia6  and  Lgmn  expression (relative the  Hprt  housekeeping gene) in CD4 +  mature thymocytes sorted from wild-type (WT) mice, CD8 +  mature thymocytes sorted from  H2ab1 −/−  or  Tcf7 −/− Lef1 −/− H2ab1 −/−  mice. ( e ) Intracellular staining for Foxp3 and Rorγt proteins in CD8 +  mature thymocytes in  H2ab1 −/−  mice, CD8 + CD4 −  and CD8 + CD4 +  thymocytes in  Tcf7 −/− Lef1 −/− H2ab1 −/−  mice. Numbers adjacent to outlined areas indicate percent Foxp3 +  and Rorγt +  cells in lower panels. Data are from one experiment measuring duplicate samples ( a-c ), from 2 experiments ( d , means ± s.d., n ≥ 4), or representative of ≥ 5 experiments ( e ).
    Figure Legend Snippet: Tcf1 and Lef1 deficiency perturbs CD8 + T cell integrity (a) RNA-Seq analysis of genes upregulated (red) or downregulated (blue) in TCRβ hi CD24 − CD69 − CD8 + mature thymocytes sorted from Tcf7 −/− Tcf7 −/− Lef1 −/− , and control littermates, with the average FPKM values of two replicates of control (Ctrl) vs. Tcf7 −/− Lef1 −/− CD8 + T cells shown in a scatterplot, where the green lines denote gene expression changes of ≥2 fold. (b) Select upregulated genes (right margin) in Tcf7 −/− Lef1 −/− CD8 + mature thymocytes relative to control and Tcf7 −/− CD8 + thymocytes as shown in a heatmap. (c) GSEA showing enriched expression of genes in the CD4 + T cell gene set in Tcf7 −/− Lef1 −/− CD8 + mature thymocytes, with the enriched genes (red rectangle in the enrichment plot) displayed in a heatmap, where CD4 + signature genes are highlighted in green. (d) Quantitative RT-PCR analysis of Cd40lg, St8sia6 and Lgmn expression (relative the Hprt housekeeping gene) in CD4 + mature thymocytes sorted from wild-type (WT) mice, CD8 + mature thymocytes sorted from H2ab1 −/− or Tcf7 −/− Lef1 −/− H2ab1 −/− mice. ( e ) Intracellular staining for Foxp3 and Rorγt proteins in CD8 + mature thymocytes in H2ab1 −/− mice, CD8 + CD4 − and CD8 + CD4 + thymocytes in Tcf7 −/− Lef1 −/− H2ab1 −/− mice. Numbers adjacent to outlined areas indicate percent Foxp3 + and Rorγt + cells in lower panels. Data are from one experiment measuring duplicate samples ( a-c ), from 2 experiments ( d , means ± s.d., n ≥ 4), or representative of ≥ 5 experiments ( e ).

    Techniques Used: RNA Sequencing Assay, Expressing, Quantitative RT-PCR, Mouse Assay, Staining

    3) Product Images from "DNMT3A and TET2 compete and cooperate to repress lineage-specific transcription factors in hematopoietic stem cells"

    Article Title: DNMT3A and TET2 compete and cooperate to repress lineage-specific transcription factors in hematopoietic stem cells

    Journal: Nature genetics

    doi: 10.1038/ng.3610

    Hydroxymethylation (hmC) in HSCs is associated with active HSC genes and repressed RBC genes a ) Numbers of hypermethylated and hypomethylated DhMRs in HSCs of Dnmt3a −/− , Tet2 −/− and DKO phenotype. b ) Heat map depicting differentially hydroxylmethylated regions (DhMRs) of the 5 major dynamic DNA hydroxylmethylation alteration patterns (DhMAPs). c ) Cytosine-5-methylenesulfonate (CMS) signal in HSCs displayed for DMR regions. d ) CMS hmC signal in displayed in relationship to genic features. e–h ) Differential CMS signal distribution in regions within the 6 major DMR classes in HSCs.
    Figure Legend Snippet: Hydroxymethylation (hmC) in HSCs is associated with active HSC genes and repressed RBC genes a ) Numbers of hypermethylated and hypomethylated DhMRs in HSCs of Dnmt3a −/− , Tet2 −/− and DKO phenotype. b ) Heat map depicting differentially hydroxylmethylated regions (DhMRs) of the 5 major dynamic DNA hydroxylmethylation alteration patterns (DhMAPs). c ) Cytosine-5-methylenesulfonate (CMS) signal in HSCs displayed for DMR regions. d ) CMS hmC signal in displayed in relationship to genic features. e–h ) Differential CMS signal distribution in regions within the 6 major DMR classes in HSCs.

    Techniques Used:

    DNA methylation across the genotypes are highly dynamic a) Flowchart of DNA methylation analysis strategy. b) Heat map depicting differentially methylated regions (DMRs) of the 6 major dynamic DNA methylation alteration patterns (DMAPs). c) Numbers of hypermethylated and hypomethylated DMRs in HSCs of Dnmt3a −/− , Tet2 −/− and DKO phenotype. d) Global DNA methylation levels of all DMRs in all 4 genotypes. Each group of genotype consisted of 2 biological replicates. e) DNA methylation levels in hematopoiesis-associated enhancers in different lineages and progenitors. Enhancers (left panel) as defined by H3K27Ac marks in B cell, RBC progenitors, ST- and LT-HSCs (from ref. 43 ), and (right panel) their DNA methylation levels.
    Figure Legend Snippet: DNA methylation across the genotypes are highly dynamic a) Flowchart of DNA methylation analysis strategy. b) Heat map depicting differentially methylated regions (DMRs) of the 6 major dynamic DNA methylation alteration patterns (DMAPs). c) Numbers of hypermethylated and hypomethylated DMRs in HSCs of Dnmt3a −/− , Tet2 −/− and DKO phenotype. d) Global DNA methylation levels of all DMRs in all 4 genotypes. Each group of genotype consisted of 2 biological replicates. e) DNA methylation levels in hematopoiesis-associated enhancers in different lineages and progenitors. Enhancers (left panel) as defined by H3K27Ac marks in B cell, RBC progenitors, ST- and LT-HSCs (from ref. 43 ), and (right panel) their DNA methylation levels.

    Techniques Used: DNA Methylation Assay, Methylation

    4) Product Images from "Assessment of a targeted resequencing assay as a support tool in the diagnosis of lysosomal storage disorders"

    Article Title: Assessment of a targeted resequencing assay as a support tool in the diagnosis of lysosomal storage disorders

    Journal: Orphanet Journal of Rare Diseases

    doi: 10.1186/1750-1172-9-59

    Global flowchart of the filtering pipeline used for selection of most likely pathogenic mutations, starting from SOLiD4 raw data. (NRA: non-reference allele).
    Figure Legend Snippet: Global flowchart of the filtering pipeline used for selection of most likely pathogenic mutations, starting from SOLiD4 raw data. (NRA: non-reference allele).

    Techniques Used: Selection

    5) Product Images from "Copy number analysis by low coverage whole genome sequencing using ultra low-input DNA from formalin-fixed paraffin embedded tumor tissue"

    Article Title: Copy number analysis by low coverage whole genome sequencing using ultra low-input DNA from formalin-fixed paraffin embedded tumor tissue

    Journal: Genome Medicine

    doi: 10.1186/s13073-016-0375-z

    CN profiles of unamplified (UA) and WGA samples. a CN profile of MCC sample MCT4, which is unamplified and untreated, with 100 ng input of DNA. b CN profile of the same sample, which is unamplified and untreated, down-sampled to the similar coverage as the matched WGA sample. c CN profile of the same sample, which is WGA and untreated. Each point represents the normalized read count ratio of a 50 kb sized bin. Separate chromosomes from 1 to 22 as well as X and Y are shown and a log 2 (copy number/2) equal to zero corresponds to a copy number of 2. Segments were removed from highly repetitive or problematic regions [ 8 ]
    Figure Legend Snippet: CN profiles of unamplified (UA) and WGA samples. a CN profile of MCC sample MCT4, which is unamplified and untreated, with 100 ng input of DNA. b CN profile of the same sample, which is unamplified and untreated, down-sampled to the similar coverage as the matched WGA sample. c CN profile of the same sample, which is WGA and untreated. Each point represents the normalized read count ratio of a 50 kb sized bin. Separate chromosomes from 1 to 22 as well as X and Y are shown and a log 2 (copy number/2) equal to zero corresponds to a copy number of 2. Segments were removed from highly repetitive or problematic regions [ 8 ]

    Techniques Used: Whole Genome Amplification

    Experimental design testing a low-input method (NEBNext Ultra II) on copy number detection of two FFPE MCC samples by LC WGS as well as the effect of WGA with or without NEB DNA repair treatment
    Figure Legend Snippet: Experimental design testing a low-input method (NEBNext Ultra II) on copy number detection of two FFPE MCC samples by LC WGS as well as the effect of WGA with or without NEB DNA repair treatment

    Techniques Used: Formalin-fixed Paraffin-Embedded, Whole Genome Amplification

    6) Product Images from "Copy number analysis by low coverage whole genome sequencing using ultra low-input DNA from formalin-fixed paraffin embedded tumor tissue"

    Article Title: Copy number analysis by low coverage whole genome sequencing using ultra low-input DNA from formalin-fixed paraffin embedded tumor tissue

    Journal: Genome Medicine

    doi: 10.1186/s13073-016-0375-z

    CN profiles of unamplified (UA) and WGA samples. a CN profile of MCC sample MCT4, which is unamplified and untreated, with 100 ng input of DNA. b CN profile of the same sample, which is unamplified and untreated, down-sampled to the similar coverage as the matched WGA sample. c CN profile of the same sample, which is WGA and untreated. Each point represents the normalized read count ratio of a 50 kb sized bin. Separate chromosomes from 1 to 22 as well as X and Y are shown and a log 2 (copy number/2) equal to zero corresponds to a copy number of 2. Segments were removed from highly repetitive or problematic regions [ 8 ]
    Figure Legend Snippet: CN profiles of unamplified (UA) and WGA samples. a CN profile of MCC sample MCT4, which is unamplified and untreated, with 100 ng input of DNA. b CN profile of the same sample, which is unamplified and untreated, down-sampled to the similar coverage as the matched WGA sample. c CN profile of the same sample, which is WGA and untreated. Each point represents the normalized read count ratio of a 50 kb sized bin. Separate chromosomes from 1 to 22 as well as X and Y are shown and a log 2 (copy number/2) equal to zero corresponds to a copy number of 2. Segments were removed from highly repetitive or problematic regions [ 8 ]

    Techniques Used: Whole Genome Amplification

    Experimental design testing a low-input method (NEBNext Ultra II) on copy number detection of two FFPE MCC samples by LC WGS as well as the effect of WGA with or without NEB DNA repair treatment
    Figure Legend Snippet: Experimental design testing a low-input method (NEBNext Ultra II) on copy number detection of two FFPE MCC samples by LC WGS as well as the effect of WGA with or without NEB DNA repair treatment

    Techniques Used: Formalin-fixed Paraffin-Embedded, Whole Genome Amplification

    7) Product Images from "LEF-1 and TCF-1 orchestrate T follicular helper cell differentiation by regulating differentiation circuits upstream of Bcl6"

    Article Title: LEF-1 and TCF-1 orchestrate T follicular helper cell differentiation by regulating differentiation circuits upstream of Bcl6

    Journal: Nature immunology

    doi: 10.1038/ni.3226

    Enhanced  Lef1  expression leads to augmented T FH  differentiation (a)  Expression of LEF-1 in GFP-RV +  (blue) and  Lef1 -RV +  (red) SMARTA cells assessed by flow cytometry.  (b-g)  Frequencies and phenotypes of GFP-RV +  or  Lef1 -RV +  SMARTA cells (CD45.1 + CD4 + CD19 − ) assessed by flow cytometry at 8 d after SMARTA cell transfer into B6 mice (CD45.2 + ) and LCMV infection.  (b)  Abundance of RV +  SMARTA cell (GFP + CD45.1 + CD4 + CD19 − ) among total CD4 +  T cells.  (c)  Abundance of SLAM lo CXCR5 +  T FH  cells among RV +  SMARTA cells.  (d-e)  Expression of canonical T FH  markers CXCR5  (d)  and PD-1  (e)  on CXCR5 −  T H 1 and CXCR5 +  T FH  cells by  Lef1 -RV +  and GFP-RV +  cells, normalized to the mean MFI per group (mean ± s.e.m.).  (f-g)  Abundance of GC T FH  cells phenotyped as PSGL-1 lo CXCR5 + (f)  and PD-1 hi CXCR5 + (e)  of RV +  SMARTA cells. Data are a composite of two independent experiments (n = 9 per group). *  P
    Figure Legend Snippet: Enhanced Lef1 expression leads to augmented T FH differentiation (a) Expression of LEF-1 in GFP-RV + (blue) and Lef1 -RV + (red) SMARTA cells assessed by flow cytometry. (b-g) Frequencies and phenotypes of GFP-RV + or Lef1 -RV + SMARTA cells (CD45.1 + CD4 + CD19 − ) assessed by flow cytometry at 8 d after SMARTA cell transfer into B6 mice (CD45.2 + ) and LCMV infection. (b) Abundance of RV + SMARTA cell (GFP + CD45.1 + CD4 + CD19 − ) among total CD4 + T cells. (c) Abundance of SLAM lo CXCR5 + T FH cells among RV + SMARTA cells. (d-e) Expression of canonical T FH markers CXCR5 (d) and PD-1 (e) on CXCR5 − T H 1 and CXCR5 + T FH cells by Lef1 -RV + and GFP-RV + cells, normalized to the mean MFI per group (mean ± s.e.m.). (f-g) Abundance of GC T FH cells phenotyped as PSGL-1 lo CXCR5 + (f) and PD-1 hi CXCR5 + (e) of RV + SMARTA cells. Data are a composite of two independent experiments (n = 9 per group). * P

    Techniques Used: Expressing, Flow Cytometry, Cytometry, Mouse Assay, Infection

    TCF-1 binds to key T FH -associated genes in T FH  cells (a-d)  ChIP assays using anti-TCF-1 antibody or control IgG were performed on naive control CD4 +  T cells (CD44 lo CD62L + CD4 + ); naive  Tcf7 −/−  CD4 +  T cells (GFP + CD44 lo CD62L + CD4 + ); WT T FH  cells (CXCR5 + CD44 hi CD62L − CD4 + ); and WT T H 1 cells (CXCR5 − CD44 hi CD62L − CD4 + ). T FH  and T H 1 cells were sorted from B6 mice 8 d after vaccinia virus infection. Quantitation of enriched TCF-1 binding was done at the positive control  Axin2  gene  (a) , the TSS of the  Il6ra  and  Il6st  genes  (b) , the TSS and a −2.8 kb upstream regulatory region of the  Bcl6  gene  (c) , and the TSS of  Ascl2  and intron 3 of  Prdm1 (d) , and data are means ± s.d. from 3 independent experiments. *  P
    Figure Legend Snippet: TCF-1 binds to key T FH -associated genes in T FH cells (a-d) ChIP assays using anti-TCF-1 antibody or control IgG were performed on naive control CD4 + T cells (CD44 lo CD62L + CD4 + ); naive Tcf7 −/− CD4 + T cells (GFP + CD44 lo CD62L + CD4 + ); WT T FH cells (CXCR5 + CD44 hi CD62L − CD4 + ); and WT T H 1 cells (CXCR5 − CD44 hi CD62L − CD4 + ). T FH and T H 1 cells were sorted from B6 mice 8 d after vaccinia virus infection. Quantitation of enriched TCF-1 binding was done at the positive control Axin2 gene (a) , the TSS of the Il6ra and Il6st genes (b) , the TSS and a −2.8 kb upstream regulatory region of the Bcl6 gene (c) , and the TSS of Ascl2 and intron 3 of Prdm1 (d) , and data are means ± s.d. from 3 independent experiments. * P

    Techniques Used: Chromatin Immunoprecipitation, Mouse Assay, Infection, Quantitation Assay, Binding Assay, Positive Control

    LEF-1-dependent T FH  differentiation supports germinal center responses ( a-e ) Frequencies and phenotypes of sh Ctrl +  or sh Lef1 +  CD45.1 +  SMARTA cells assessed by flow cytometry at 8 d after SMARTA cell transfer into B6 mice and LCMV infection.  (a)  Abundance of shRNA +  SMARTA cells (Ametrine + CD45.1 + CD4 + CD19 − ) among total CD4 +  T cells.  (b)  Frequency of SLAM lo CXCR5 +  T FH  cells. Quantitation shown as % of SMARTA cells.  (c)  Expression of CXCR5 on sh Ctrl +  (blue) and sh Lef1 +  (red) SMARTA cells.  (d-e)  Frequencies of sh Ctrl +  and sh Lef1 +  SMARTA GC T FH  cells among SMARTA cells phenotyped as PSGL-1 lo CXCR5 + (d)  and Bcl6 hi CXCR5 +  ( e ).  (f)  Abundance of GC B cells (Bcl6 + CD19 + ) among total B cells. Data are representative of two independent experiments (n = 4–5 per group, mean ± s.e.m.). *  P
    Figure Legend Snippet: LEF-1-dependent T FH differentiation supports germinal center responses ( a-e ) Frequencies and phenotypes of sh Ctrl + or sh Lef1 + CD45.1 + SMARTA cells assessed by flow cytometry at 8 d after SMARTA cell transfer into B6 mice and LCMV infection. (a) Abundance of shRNA + SMARTA cells (Ametrine + CD45.1 + CD4 + CD19 − ) among total CD4 + T cells. (b) Frequency of SLAM lo CXCR5 + T FH cells. Quantitation shown as % of SMARTA cells. (c) Expression of CXCR5 on sh Ctrl + (blue) and sh Lef1 + (red) SMARTA cells. (d-e) Frequencies of sh Ctrl + and sh Lef1 + SMARTA GC T FH cells among SMARTA cells phenotyped as PSGL-1 lo CXCR5 + (d) and Bcl6 hi CXCR5 + ( e ). (f) Abundance of GC B cells (Bcl6 + CD19 + ) among total B cells. Data are representative of two independent experiments (n = 4–5 per group, mean ± s.e.m.). * P

    Techniques Used: Flow Cytometry, Cytometry, Mouse Assay, Infection, shRNA, Quantitation Assay, Expressing

    Genetic ablation of LEF-1 impairs GC T FH  differentiation ( a-c ) Flow cytometry of T FH  and GC T FH  cells in spleens of  Lef1 −/−  mice and littermate controls infecte d w ith vaccinia virus for 8 days. Plots are gated on CD44 hi CD62L lo GFP + CD4 +  T cells.  (a)  SLAM lo CXCR5 +  T FH  cells. ( b-c ) Abundance of GC T FH  cells phenotyped as Bcl6 + CXCR5 +  ( b ) and PD-1 hi CXCR5 + (c) . Cumulative data from four independent experiments are shown (mean ± s.d.). *  P
    Figure Legend Snippet: Genetic ablation of LEF-1 impairs GC T FH differentiation ( a-c ) Flow cytometry of T FH and GC T FH cells in spleens of Lef1 −/− mice and littermate controls infecte d w ith vaccinia virus for 8 days. Plots are gated on CD44 hi CD62L lo GFP + CD4 + T cells. (a) SLAM lo CXCR5 + T FH cells. ( b-c ) Abundance of GC T FH cells phenotyped as Bcl6 + CXCR5 + ( b ) and PD-1 hi CXCR5 + (c) . Cumulative data from four independent experiments are shown (mean ± s.d.). * P

    Techniques Used: Flow Cytometry, Cytometry, Mouse Assay

    Both TCF-1 and LEF-1 contribute to regulation of T FH  differentiation and B cell responses (a)  Flow cytometry of  Tcf7 -GFP expression in SMARTA CD4 +  T cells (CD45.2 + CD4 + ) at 8 d after  Tcf7 GFP/+  SMARTA cell transfer into CD45.1 +  recipients and LCMV infection. Gated populations of T H 1 (CXCR5 − SLAM hi ) and T FH  (CXCR5 + SLAM lo ) cells were analyzed for  Tcf7 -GFP expression. Numbers indicate percent  Tcf7 -GFP +  cells. Data are representative of ≥ 2 experiments.  (b-f)  Flow cytometry of  Tcf7 −/− Lef1 −/− Tcf7 −/− , and littermate controls 8 d after infection  i.v . with vaccinia virus.  (b-d)  Abundance of SLAM lo CXCR5 +  T FH  cells  (b) , Bcl6 + CXCR5 +  GC T FH  cells  (c) , and PD-1 hi CXCR5 +  GC T FH  cells  (d)  gated on CD44 hi CD62L − GFP + CD4 +  T cells in spleen. ( e-f ) Abundance of GL7 + Fas +  GC B cells  (e)  and IgD lo CD138 +  plasma cells  (f)  in the same animals (mean ± s.d.). Cumulative data from ≥ 3 experiments are shown. *  P
    Figure Legend Snippet: Both TCF-1 and LEF-1 contribute to regulation of T FH differentiation and B cell responses (a) Flow cytometry of Tcf7 -GFP expression in SMARTA CD4 + T cells (CD45.2 + CD4 + ) at 8 d after Tcf7 GFP/+ SMARTA cell transfer into CD45.1 + recipients and LCMV infection. Gated populations of T H 1 (CXCR5 − SLAM hi ) and T FH (CXCR5 + SLAM lo ) cells were analyzed for Tcf7 -GFP expression. Numbers indicate percent Tcf7 -GFP + cells. Data are representative of ≥ 2 experiments. (b-f) Flow cytometry of Tcf7 −/− Lef1 −/− Tcf7 −/− , and littermate controls 8 d after infection i.v . with vaccinia virus. (b-d) Abundance of SLAM lo CXCR5 + T FH cells (b) , Bcl6 + CXCR5 + GC T FH cells (c) , and PD-1 hi CXCR5 + GC T FH cells (d) gated on CD44 hi CD62L − GFP + CD4 + T cells in spleen. ( e-f ) Abundance of GL7 + Fas + GC B cells (e) and IgD lo CD138 + plasma cells (f) in the same animals (mean ± s.d.). Cumulative data from ≥ 3 experiments are shown. * P

    Techniques Used: Flow Cytometry, Cytometry, Expressing, Infection

    Lef1  expression is associated with T FH  cells and regulates early T FH  differentiation (a)  RNA-seq analysis of early T FH  (IL-2Rα − Blimp1-YFP − ) versus T H 1 (IL-2Rα + Blimp1-YFP + ) CD45.1 +  Blimp1-YFP SMARTA cells isolated from B6 mice 3 d after SMARTA cell transfer and LCMV infection (left panels), and that of T H 1 (CXCR5 − ), T FH  (PD-1 lo CXCR5 + ), and GC T FH  (PD-1 hi CXCR5 + ) sorted 8 d after LCMV from CD45.2 +  B6 mice (right panels). Heatmaps of selected genes of interest are shown.  (b)  Scatter plot of genes showing ≥ |1.5 fold| differential expression in early T FH  in comparison to T H 1 cells. Select genes of interest are marked.  (c)  Immunoblot of LEF-1 (two isoforms) and β-actin from sh Ctrl +  and sh Lef1 +  SMARTA cells.  (d-f)  Analysis of sh Ctrl +  or sh Lef1 +  CD45.1 +  SMARTA cells (Ametrine + CD45.1 + CD4 + CD19 − ), three days after transfer of shRNA-RV-infected SMARTA cells into B6 mice and LCMV infection.  (d)  shRNA +  SMARTA cell frequency among total CD4 +  T cells.  (e-f)  Phenotyping of sh Ctrl +  and sh Lef1 +  SMARTA cells. ( e ) Bcl6 + CXCR5 +  T FH  cells.  (f)  IL-2Rα − CXCR5 +  T FH  cells. Quantitation shown as % of SMARTA cells (mean ± s.e.m.). Data are a composite of two independent experiments (n = 7 per group). *  P
    Figure Legend Snippet: Lef1 expression is associated with T FH cells and regulates early T FH differentiation (a) RNA-seq analysis of early T FH (IL-2Rα − Blimp1-YFP − ) versus T H 1 (IL-2Rα + Blimp1-YFP + ) CD45.1 + Blimp1-YFP SMARTA cells isolated from B6 mice 3 d after SMARTA cell transfer and LCMV infection (left panels), and that of T H 1 (CXCR5 − ), T FH (PD-1 lo CXCR5 + ), and GC T FH (PD-1 hi CXCR5 + ) sorted 8 d after LCMV from CD45.2 + B6 mice (right panels). Heatmaps of selected genes of interest are shown. (b) Scatter plot of genes showing ≥ |1.5 fold| differential expression in early T FH in comparison to T H 1 cells. Select genes of interest are marked. (c) Immunoblot of LEF-1 (two isoforms) and β-actin from sh Ctrl + and sh Lef1 + SMARTA cells. (d-f) Analysis of sh Ctrl + or sh Lef1 + CD45.1 + SMARTA cells (Ametrine + CD45.1 + CD4 + CD19 − ), three days after transfer of shRNA-RV-infected SMARTA cells into B6 mice and LCMV infection. (d) shRNA + SMARTA cell frequency among total CD4 + T cells. (e-f) Phenotyping of sh Ctrl + and sh Lef1 + SMARTA cells. ( e ) Bcl6 + CXCR5 + T FH cells. (f) IL-2Rα − CXCR5 + T FH cells. Quantitation shown as % of SMARTA cells (mean ± s.e.m.). Data are a composite of two independent experiments (n = 7 per group). * P

    Techniques Used: Expressing, RNA Sequencing Assay, Isolation, Mouse Assay, Infection, shRNA, Quantitation Assay

    8) Product Images from "RNA sequencing of the nephron transcriptome: a technical note"

    Article Title: RNA sequencing of the nephron transcriptome: a technical note

    Journal: Kidney Research and Clinical Practice

    doi: 10.1016/j.krcp.2015.08.008

    The workflow of the RNA-seq profiling of the nephron transcriptome. Poly(A)′-mRNAs released from microdissected renal tubule segments are prepared into adapter-ligated cDNA libraries through reverse transcription and amplification. Illumina sequencing generates 50-bp paired-end FASTQ sequences. cDNAs, complementary DNAs; poly(A)′-mRNA, polyadenylated messenger RNA; RNA-seq, RNA sequencing.
    Figure Legend Snippet: The workflow of the RNA-seq profiling of the nephron transcriptome. Poly(A)′-mRNAs released from microdissected renal tubule segments are prepared into adapter-ligated cDNA libraries through reverse transcription and amplification. Illumina sequencing generates 50-bp paired-end FASTQ sequences. cDNAs, complementary DNAs; poly(A)′-mRNA, polyadenylated messenger RNA; RNA-seq, RNA sequencing.

    Techniques Used: RNA Sequencing Assay, Amplification, Sequencing

    9) Product Images from "Large conserved domains of low DNA methylation maintained by Dnmt3a"

    Article Title: Large conserved domains of low DNA methylation maintained by Dnmt3a

    Journal: Nature genetics

    doi: 10.1038/ng.2836

    Large undermethylated Canyons revealed by WGBS ( a ) UCSC genome browser track depicts methylation profile across the Pax6 gene in murine HSCs. Methylation ratios from 0% to 100%, for individual CpG sites are shown in red. The identified Undermethylated regions (UMRs) (≤10% methylation) are indicated by blue bars, while the CpG islands are indicated in green, repeats are marked in black, and mammalian conservation is shown in dark blue. RNA-seq expression is shown at bottom in green (the Pax6 promoter is in the center of the Canyon and has no RNAseq signal; the signal on the right of the plot comes from the 3’ end of the adjacent gene which is transcribed toward Pax6 ). ( b ) Gene ontology analysis of Canyon-associated genes. Ontology terms are shown on the y-axis; p-value for each category based on functional studies is graphed along the x-axis. ( c ) Overlap of four gene groups 15 using different UMR-length cutoffs. GRB targets genes are predicted regulatory targets of the highly conserved non-coding elements in genomic regulatory blocks (GRBs). Bystander genes are contained within GRBs but under distinct control. Other CGI genes overlap with CGI, but are not associated with GRBs, and the Other TF genes are transcription factors not associated with GRBs. The x-axis indicates the length cutoff of UMRs and y-axis indicates the percent of UMR-overlapping genes relative to all genes in each respective group. ( d ) The proportion of Canyons that contain the indicated numbers of CGIs. ( e ) Position of binding peaks for 10 TFs (SCL/TAL1, LYL1, LMO2, GATA2, RUNX1, MEIS1, PU.1, ERG, FLI-1 , and GFI1B ) across Canyons. The normalized Canyons are indicated with a position of 0 representing the Canyon centers, and positions ±1 representing the Canyon edges, as indicated by the blue bar. ( f ) Pax7 -asscociated Canyons in human cells; data from the human Epigenome Atlas project. CD34: Mobilized CD34+ primary cells, Liver: Adult Liver, Brain: Brain Germinal Matrix, NGED: Neurosphere cultured cells– Ganglionic Eminence Derived, MSC: Human ES cell (H1)–derived Mesenchymal stem cells, NPC: H1-derived Neuronal Progenitor cultured cells, Human ES cells (H9), CD184: CD184+ Endoderm cultured cells, iPS: iPS DF 6.9 cell line, BHM: Brain Hippocampus Middle. Data obtained from NIH Roadmap Epigenomics Mapping Consortium ( www.roadmapepigenomics.org ).
    Figure Legend Snippet: Large undermethylated Canyons revealed by WGBS ( a ) UCSC genome browser track depicts methylation profile across the Pax6 gene in murine HSCs. Methylation ratios from 0% to 100%, for individual CpG sites are shown in red. The identified Undermethylated regions (UMRs) (≤10% methylation) are indicated by blue bars, while the CpG islands are indicated in green, repeats are marked in black, and mammalian conservation is shown in dark blue. RNA-seq expression is shown at bottom in green (the Pax6 promoter is in the center of the Canyon and has no RNAseq signal; the signal on the right of the plot comes from the 3’ end of the adjacent gene which is transcribed toward Pax6 ). ( b ) Gene ontology analysis of Canyon-associated genes. Ontology terms are shown on the y-axis; p-value for each category based on functional studies is graphed along the x-axis. ( c ) Overlap of four gene groups 15 using different UMR-length cutoffs. GRB targets genes are predicted regulatory targets of the highly conserved non-coding elements in genomic regulatory blocks (GRBs). Bystander genes are contained within GRBs but under distinct control. Other CGI genes overlap with CGI, but are not associated with GRBs, and the Other TF genes are transcription factors not associated with GRBs. The x-axis indicates the length cutoff of UMRs and y-axis indicates the percent of UMR-overlapping genes relative to all genes in each respective group. ( d ) The proportion of Canyons that contain the indicated numbers of CGIs. ( e ) Position of binding peaks for 10 TFs (SCL/TAL1, LYL1, LMO2, GATA2, RUNX1, MEIS1, PU.1, ERG, FLI-1 , and GFI1B ) across Canyons. The normalized Canyons are indicated with a position of 0 representing the Canyon centers, and positions ±1 representing the Canyon edges, as indicated by the blue bar. ( f ) Pax7 -asscociated Canyons in human cells; data from the human Epigenome Atlas project. CD34: Mobilized CD34+ primary cells, Liver: Adult Liver, Brain: Brain Germinal Matrix, NGED: Neurosphere cultured cells– Ganglionic Eminence Derived, MSC: Human ES cell (H1)–derived Mesenchymal stem cells, NPC: H1-derived Neuronal Progenitor cultured cells, Human ES cells (H9), CD184: CD184+ Endoderm cultured cells, iPS: iPS DF 6.9 cell line, BHM: Brain Hippocampus Middle. Data obtained from NIH Roadmap Epigenomics Mapping Consortium ( www.roadmapepigenomics.org ).

    Techniques Used: Methylation, RNA Sequencing Assay, Expressing, Functional Assay, Binding Assay, Cell Culture, Derivative Assay

    10) Product Images from "The histone demethylase KDM3A regulates the transcriptional program of the androgen receptor in prostate cancer cells"

    Article Title: The histone demethylase KDM3A regulates the transcriptional program of the androgen receptor in prostate cancer cells

    Journal: Oncotarget

    doi: 10.18632/oncotarget.15681

    Detection of histone demethylase events by ChIP-qRT-PCR due to KDM3A activity in target genes involved in the androgen response CWR22Rv1 prostate cancer cells transfected with pLKO.1 control or KDM3A shRNA were subjected to the chromatin immunoprecipitation coupled with quantitative real time polymerase chain reaction (ChIP-qRT-PCR) assay using immunoprecipitation with A . H3K9me1-antibody and B . H3K9me2-antibody. The precipitated chromatin fragments were analyzed by qRT-PCR using oligonucleotides for identified androgen response element regions of KLK3 , NKX3-1 or MYC .
    Figure Legend Snippet: Detection of histone demethylase events by ChIP-qRT-PCR due to KDM3A activity in target genes involved in the androgen response CWR22Rv1 prostate cancer cells transfected with pLKO.1 control or KDM3A shRNA were subjected to the chromatin immunoprecipitation coupled with quantitative real time polymerase chain reaction (ChIP-qRT-PCR) assay using immunoprecipitation with A . H3K9me1-antibody and B . H3K9me2-antibody. The precipitated chromatin fragments were analyzed by qRT-PCR using oligonucleotides for identified androgen response element regions of KLK3 , NKX3-1 or MYC .

    Techniques Used: Chromatin Immunoprecipitation, Quantitative RT-PCR, Activity Assay, Transfection, shRNA, Real-time Polymerase Chain Reaction, Immunoprecipitation

    Related Articles

    Transfection:

    Article Title: CRISPR-Cas3 induces broad and unidirectional genome editing in human cells
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    Next-Generation Sequencing:

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    Article Snippet: .. High Throughput Sequencing (HTS) Eight micrograms of total genomic DNA of Plasmodium chabaudi chabaudi isolate CB (Pcc CB) obtained from the blood of infected mice was fragmented in a microTUBE with an Adaptive Focused Acoustics fiber (Covaris, Woburn, Massachusetts) using an S2 focused ultra-sonicator (Covaris) set at 5% duty cycle; intensity 4; 200 cycles/burst for 90 s. In order to minimize the undesirable bias that affects the sequence analysis of extremely A/T-rich DNA such as that of Plasmodium spp., we processed the fragmented DNA using a NEXTflex PCR-Free DNA Sequencing Kit (BIOO Scientific, Austin, Texas) and NEXTflex PCR-Free barcode 1 (BIOO Scientific) following the protocol provided with the kit. .. The processed DNA was applied to an agarose gel on E-Gel iBase (Life Technologies, Carlsbad, California) and fragments that ran as 400–500 bp were collected to form the sequence library for further analysis.

    Infection:

    Article Title: The Unique Structure of the Apicoplast Genome of the Rodent Malaria Parasite Plasmodium chabaudi chabaudi
    Article Snippet: .. High Throughput Sequencing (HTS) Eight micrograms of total genomic DNA of Plasmodium chabaudi chabaudi isolate CB (Pcc CB) obtained from the blood of infected mice was fragmented in a microTUBE with an Adaptive Focused Acoustics fiber (Covaris, Woburn, Massachusetts) using an S2 focused ultra-sonicator (Covaris) set at 5% duty cycle; intensity 4; 200 cycles/burst for 90 s. In order to minimize the undesirable bias that affects the sequence analysis of extremely A/T-rich DNA such as that of Plasmodium spp., we processed the fragmented DNA using a NEXTflex PCR-Free DNA Sequencing Kit (BIOO Scientific, Austin, Texas) and NEXTflex PCR-Free barcode 1 (BIOO Scientific) following the protocol provided with the kit. .. The processed DNA was applied to an agarose gel on E-Gel iBase (Life Technologies, Carlsbad, California) and fragments that ran as 400–500 bp were collected to form the sequence library for further analysis.

    Mouse Assay:

    Article Title: The Unique Structure of the Apicoplast Genome of the Rodent Malaria Parasite Plasmodium chabaudi chabaudi
    Article Snippet: .. High Throughput Sequencing (HTS) Eight micrograms of total genomic DNA of Plasmodium chabaudi chabaudi isolate CB (Pcc CB) obtained from the blood of infected mice was fragmented in a microTUBE with an Adaptive Focused Acoustics fiber (Covaris, Woburn, Massachusetts) using an S2 focused ultra-sonicator (Covaris) set at 5% duty cycle; intensity 4; 200 cycles/burst for 90 s. In order to minimize the undesirable bias that affects the sequence analysis of extremely A/T-rich DNA such as that of Plasmodium spp., we processed the fragmented DNA using a NEXTflex PCR-Free DNA Sequencing Kit (BIOO Scientific, Austin, Texas) and NEXTflex PCR-Free barcode 1 (BIOO Scientific) following the protocol provided with the kit. .. The processed DNA was applied to an agarose gel on E-Gel iBase (Life Technologies, Carlsbad, California) and fragments that ran as 400–500 bp were collected to form the sequence library for further analysis.

    Real-time Polymerase Chain Reaction:

    Article Title: Tcf1 and Lef1 transcription factors establish CD8+ T cell identity through intrinsic HDAC activity
    Article Snippet: .. ChIP and quantitative PCR WT CD4+ or CD8+ , Tcf7 −/− Lef1 −/− CD8+ mature thymocytes or splenic CD8+ T cells were sorted and cross-linked with 1% formaldehyde in medium for 5 minutes, processed using truChIP Chromatin Shearing Reagent Kit (Covaris), and sonicated for 5 minutes on Covaris S2 ultrasonicator. .. The resulting sheared chromatin fragments were in 200–500 bp range and were immunoprecipitated with anti-H3K4me3 (Millipore, 17–614), H3K9Ac (Abcam, ab4441), H3K27me3 (Millipore, 17–622), H3K27Ac (Abcam, ab4729), or control IgG and washed as previously described .

    Sequencing:

    Article Title: The Unique Structure of the Apicoplast Genome of the Rodent Malaria Parasite Plasmodium chabaudi chabaudi
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    DNA Sequencing:

    Article Title: The Unique Structure of the Apicoplast Genome of the Rodent Malaria Parasite Plasmodium chabaudi chabaudi
    Article Snippet: .. High Throughput Sequencing (HTS) Eight micrograms of total genomic DNA of Plasmodium chabaudi chabaudi isolate CB (Pcc CB) obtained from the blood of infected mice was fragmented in a microTUBE with an Adaptive Focused Acoustics fiber (Covaris, Woburn, Massachusetts) using an S2 focused ultra-sonicator (Covaris) set at 5% duty cycle; intensity 4; 200 cycles/burst for 90 s. In order to minimize the undesirable bias that affects the sequence analysis of extremely A/T-rich DNA such as that of Plasmodium spp., we processed the fragmented DNA using a NEXTflex PCR-Free DNA Sequencing Kit (BIOO Scientific, Austin, Texas) and NEXTflex PCR-Free barcode 1 (BIOO Scientific) following the protocol provided with the kit. .. The processed DNA was applied to an agarose gel on E-Gel iBase (Life Technologies, Carlsbad, California) and fragments that ran as 400–500 bp were collected to form the sequence library for further analysis.

    Periodic Counter-current Chromatography:

    Article Title: The Unique Structure of the Apicoplast Genome of the Rodent Malaria Parasite Plasmodium chabaudi chabaudi
    Article Snippet: .. High Throughput Sequencing (HTS) Eight micrograms of total genomic DNA of Plasmodium chabaudi chabaudi isolate CB (Pcc CB) obtained from the blood of infected mice was fragmented in a microTUBE with an Adaptive Focused Acoustics fiber (Covaris, Woburn, Massachusetts) using an S2 focused ultra-sonicator (Covaris) set at 5% duty cycle; intensity 4; 200 cycles/burst for 90 s. In order to minimize the undesirable bias that affects the sequence analysis of extremely A/T-rich DNA such as that of Plasmodium spp., we processed the fragmented DNA using a NEXTflex PCR-Free DNA Sequencing Kit (BIOO Scientific, Austin, Texas) and NEXTflex PCR-Free barcode 1 (BIOO Scientific) following the protocol provided with the kit. .. The processed DNA was applied to an agarose gel on E-Gel iBase (Life Technologies, Carlsbad, California) and fragments that ran as 400–500 bp were collected to form the sequence library for further analysis.

    Polymerase Chain Reaction:

    Article Title: The Unique Structure of the Apicoplast Genome of the Rodent Malaria Parasite Plasmodium chabaudi chabaudi
    Article Snippet: .. High Throughput Sequencing (HTS) Eight micrograms of total genomic DNA of Plasmodium chabaudi chabaudi isolate CB (Pcc CB) obtained from the blood of infected mice was fragmented in a microTUBE with an Adaptive Focused Acoustics fiber (Covaris, Woburn, Massachusetts) using an S2 focused ultra-sonicator (Covaris) set at 5% duty cycle; intensity 4; 200 cycles/burst for 90 s. In order to minimize the undesirable bias that affects the sequence analysis of extremely A/T-rich DNA such as that of Plasmodium spp., we processed the fragmented DNA using a NEXTflex PCR-Free DNA Sequencing Kit (BIOO Scientific, Austin, Texas) and NEXTflex PCR-Free barcode 1 (BIOO Scientific) following the protocol provided with the kit. .. The processed DNA was applied to an agarose gel on E-Gel iBase (Life Technologies, Carlsbad, California) and fragments that ran as 400–500 bp were collected to form the sequence library for further analysis.

    Article Title: Advanced Whole Genome Sequencing Using a Complete PCR-free Massively Parallel Sequencing (MPS) Workflow
    Article Snippet: .. For acoustic PCR-free libraries, genomic DNA was fragmented to 100-600 bp with peak size at 350-400 bp using LE220 Ultrasonicator (Covaris, Woburn, MA, USA). .. For FS PCR-free libraries, genomic DNA was fragmented to 100-1000 bp with peak size at 350-475 bp using enzymatic shearing method.

    Sonication:

    Article Title: Tcf1 and Lef1 transcription factors establish CD8+ T cell identity through intrinsic HDAC activity
    Article Snippet: .. ChIP and quantitative PCR WT CD4+ or CD8+ , Tcf7 −/− Lef1 −/− CD8+ mature thymocytes or splenic CD8+ T cells were sorted and cross-linked with 1% formaldehyde in medium for 5 minutes, processed using truChIP Chromatin Shearing Reagent Kit (Covaris), and sonicated for 5 minutes on Covaris S2 ultrasonicator. .. The resulting sheared chromatin fragments were in 200–500 bp range and were immunoprecipitated with anti-H3K4me3 (Millipore, 17–614), H3K9Ac (Abcam, ab4441), H3K27me3 (Millipore, 17–622), H3K27Ac (Abcam, ab4729), or control IgG and washed as previously described .

    Article Title: Pluripotency factors select gene expression repertoire at Zygotic Genome Activation
    Article Snippet: .. In order to shear the chromatin to 200 bp fragments (on average), the chromatin was sonicated using the Covaris S2 sonicator (DC 20 %, Intensity 5, Cycles of Burst 200, Time = 3*40 cycles with 30 sec each (3*20 min)). .. To ensure that the sonication was successful, 30 μl of the sheared chromatin was de-crosslinked with 250 mM NaCl over night at 65 °C and then analyzed using the Agilent Expert 2100 Bioanalyzer® and Agilent high sensitivity DNA Chip kit.

    Chromatin Immunoprecipitation:

    Article Title: Tcf1 and Lef1 transcription factors establish CD8+ T cell identity through intrinsic HDAC activity
    Article Snippet: .. ChIP and quantitative PCR WT CD4+ or CD8+ , Tcf7 −/− Lef1 −/− CD8+ mature thymocytes or splenic CD8+ T cells were sorted and cross-linked with 1% formaldehyde in medium for 5 minutes, processed using truChIP Chromatin Shearing Reagent Kit (Covaris), and sonicated for 5 minutes on Covaris S2 ultrasonicator. .. The resulting sheared chromatin fragments were in 200–500 bp range and were immunoprecipitated with anti-H3K4me3 (Millipore, 17–614), H3K9Ac (Abcam, ab4441), H3K27me3 (Millipore, 17–622), H3K27Ac (Abcam, ab4729), or control IgG and washed as previously described .

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  • 92
    Covaris sonication
    Sonication, supplied by Covaris, used in various techniques. Bioz Stars score: 92/100, based on 158 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/sonication/product/Covaris
    Average 92 stars, based on 158 article reviews
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    88
    Covaris s2 sonication system
    S2 Sonication System, supplied by Covaris, used in various techniques. Bioz Stars score: 88/100, based on 8 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/s2 sonication system/product/Covaris
    Average 88 stars, based on 8 article reviews
    Price from $9.99 to $1999.99
    s2 sonication system - by Bioz Stars, 2020-08
    88/100 stars
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