Journal: Cell reports

Article Title: Single-cell sequencing reveals activation of core transcription factors in PRC2-deficient malignant peripheral nerve sheath tumor

doi: 10.1016/j.celrep.2022.111363

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: SimpleChIP ChIP-seq DNA library Prep Kit , Cell Signaling Technology , Cat# 56795.

Techniques: Control, Virus, Recombinant, Lysis, Protease Inhibitor, Western Blot, Saline, Bradford Assay, Extraction, Magnetic Beads, Multiplex Assay, Next-Generation Sequencing, Generated, Sequencing, Negative Control, Plasmid Preparation, Software, Cell Analysis, Microscopy

Journal: Blood

Article Title: Enhancer-gene rewiring in the pathogenesis of Quebec platelet disorder

doi: 10.1182/blood.2020005394

Figure Lengend Snippet: QPD results in a loss of repressive chromatin and repositions PLAU relative to a candidate megakaryocyte enhancer. (A) Genome browser view of tracks produced by ChIP-seq of H3K27ac (blue), H3K4me2 (orange), and H3K36me3 (red) in granulocytes and megakaryocytes, from QPD and control (CTL), and H3K27me3 (gray) and CTCF (green) from QPD and CTL megakaryocytes only (bottom). Each track shows merged signal from 3 biological replicates with the exception of the CTL granulocyte H3K4me2, for which only 2 biological replicates were used. ChIP-seq track values are shown as fold change over input. Arrowheads mark the position of ENHQPD. The schematic (top) describes the duplication in QPD. Dashed lines mark the boundaries of the duplicated region. Tracks were visualized using the WashU epigenome browser with the following settings: summary method, max; smoothing window, 3 pixels. (B) ChIP-seq signal from QPD megakaryocytes and granulocytes aligned to a custom chromosome 10 containing the QPD duplication. Tracks were smoothed before visualization with Sushi70 in R. Only unmapped reads and reads mapping to chromosome 10 from hg19 alignments were used.

Article Snippet: All sequencing libraries were constructed using the NEBNext ChIP-seq DNA library preparation kit with the exception of H3K27me3 and CTCF ChIP-seq libraries which used NEBNext Ultra II (cat. no. E7645L).

Techniques: Produced, ChIP-sequencing

Journal: Blood

Article Title: Enhancer-gene rewiring in the pathogenesis of Quebec platelet disorder

doi: 10.1182/blood.2020005394

Figure Lengend Snippet: ENHQPD is a conserved hematopoietic enhancer that acts in synergy with the PLAU and VCL promoters. (A) Views of human ENHQPD. H3K27ac signal from QPD megakaryocytes (blue, including zoom in view) are compared with published ChIP-seq tracks for hematopoietic transcription factors from cord blood–derived megakaryocytes44 (red), expressed as fold change over input. Teal track shows a 46-way vertebrate PhastCons score. (B) Similar views of the orthologous equivalent of ENHQPD in mouse, showing ChIP-seq tracks for corresponding transcription factors profiled in mouse CD41+ hematopoietic precursor cells.45 Black squares in panels A and B (bottom) depict ENHQPD_CONS. (C) The Tol2 reporter vector system. (D) An embryo at 24 hpf (top), showing known early-stage zebrafish hematopoietic tissues (adapted from Chen and Zon,47 with permission); in situ staining (bottom) for GFP in Tg(ENHQPD_CONS:EGFP). A representative F2 embryo at 24 hpf is shown. Embryos were fixed at 24 hpf in 4% paraformaldehyde and incubated with a digoxigenin-labeled antisense gfp probe. RNA-probe hybrids were detected by an alkaline phosphatase–conjugated antibody (anti-digoxigenin-AP and Fab fragments; 1:5000; Roche, Basel, Switzerland) that catalyzed reaction on a chromogenic substrate (NBT/BCIP; Roche). Stained embryos were cleared in 2:1 benzyl benzoate/benzyl alcohol solution and imaged under an Axio Zoom.V16 Stereoscope (Zeiss, Oberkochen, Germany). PLM, posterior lateral mesoderm; ICM, interior cell mass; PBI, posterior blood island. (E) Confocal microscopy image of the PBI in a representative Tg(gata1:dsRed) x Tg(ENHQPD_CONS:EGFP) double-transgenic embryo. At 24 hpf, embryos were mounted in 1% (w/v) low-melt agarose (Sigma-Aldrich, St. Louis, MO) and imaged under a Nikon A1R Si Point scanning confocal microscope (Nikon, Tokyo, Japan). (F) Relative luciferase activity for minimal (minP), PLAU (pPLAU), or VCL (pVCL) promoter constructs, with or without ENHQPD_CONS, assayed in K562 erythroid leukemia cells. Data points show measured values averaged from 4 technical replicates (separate wells) per construct, for 5 separate transfections. All values were normalized relative to minP in their respective cell type. Statistical analysis was performed using 1-way analysis of variance with the Tukey correction. Asterisks immediately above data points denote significance compared with minP. Error bars show standard deviation of mean. Asterisks denote significance vs minP and other select pairwise comparisons. ***P < .001; n.s., not significant. All constructs and inserted sequences are further described in “Materials and methods” and supplemental Methods.

Article Snippet: All sequencing libraries were constructed using the NEBNext ChIP-seq DNA library preparation kit with the exception of H3K27me3 and CTCF ChIP-seq libraries which used NEBNext Ultra II (cat. no. E7645L).

Techniques: ChIP-sequencing, Derivative Assay, Plasmid Preparation, In Situ, Staining, Incubation, Labeling, Confocal Microscopy, Transgenic Assay, Microscopy, Luciferase, Activity Assay, Construct, Transfection, Standard Deviation

Journal: Blood

Article Title: Enhancer-gene rewiring in the pathogenesis of Quebec platelet disorder

doi: 10.1182/blood.2020005394

Figure Lengend Snippet: The QPD duplication spans the sub-TAD boundary separating PLAU and ENHQPD. (A) Hi-C interaction matrix from K562 erythroid leukemia cells. Green tracks show K562 CTCF ChIP-seq and ChIA-PET interactions. Arrowhead marks a corner-dot feature, indicative of a looping interaction. A graphic interpretation (right) of the sub-TAD structures. Domains are color coded corresponding to structures marked on the Hi-C interaction matrix: blue, sub-TADPLAU; yellow, sub-TADVCL. CTCF sites are shown in purple. (B) Zoom-in of the highlighted region in panel A. K562 CTCF ChIP-seq ChIA-PET interactions (top) as in panel A. Blue “>” and red “<” depict forward- and reverse-oriented CTCF motifs, respectively. H3K27ac (blue) and H3K27me3 (gray) and CTCF (green) ChIP-seq tracks (bottom) from QPD and control megakaryocytes (MK), as in Figure 2. Black bar marks the region duplicated in QPD. Dashed rectangles mark the inferred inactive sub-TADPLAU (left) and active sub-TADVCL (right) domains. Arrowhead marks the position of ENHQPD. (C) Black arcs show the megakaryocyte promoter capture Hi-C interactions reported by Javierre et al.51 Only interactions with a soft-thresholded, negative log-weighted P ≥ 5 are shown.

Article Snippet: All sequencing libraries were constructed using the NEBNext ChIP-seq DNA library preparation kit with the exception of H3K27me3 and CTCF ChIP-seq libraries which used NEBNext Ultra II (cat. no. E7645L).

Techniques: Hi-C, ChIP-sequencing, ChIA Pet Assay

Journal: Nature Communications

Article Title: Oncogenic enhancers drive esophageal squamous cell carcinogenesis and metastasis

doi: 10.1038/s41467-021-24813-2

Figure Lengend Snippet: a Diagram of the experimental design for profiling the transcriptome and epigenome of adjacent normal tissues (Nor), esophageal squamous cell cancer (EC) tissues, and lymph node cancer (LNC) tissues. b Identification of the differentially distributed enhancers in Nor ( n = 10), EC ( n = 10), and LNC ( n = 8) tissues. The numbers of altered (gained or lost) promoters (Pro) or enhancers (Enh) upon the comparison of Nor with EC or LNC, or EC with LNC are presented. Rel. relative. c Heatmap of the altered enhancers across Nor ( n = 10), EC ( n = 10), and LNC ( n = 8) samples with H3K27ac enrichment signals. Six groups (G1–G6) of enhancers with H3K27ac enrichment signals with the indicated number of enhancer elements are presented. d Unsupervised hierarchical clustering analysis of 28 Nor, EC, and LNC samples’ differential enhancers shown in c . The number of patients number is also provided. e Principal component analysis of 28 Nor ( n = 10), EC ( n = 10), and LNC ( n = 8) samples using all differentially distributed promoter elements shown in Supplementary Fig. and enhancers shown in c . f Venn diagrams depicting the numbers of shared enhancers (left) and super-enhancers (right) across Nor, EC, and LNC samples. g Identification of super-enhancers in EC and LNC samples in infection plots. The top super-enhancer-associated genes are labeled. The number of shared, EC-specific, and LNC-specific super-enhancers across the three types of samples are displayed in pie charts. h Tracks of H3K27ac ChIP-seq and RNA-seq data at the EHF and CCND1 loci in four representatives paired Nor, EC, and LNC samples. The previously identified enhancer upstream of the CCND1 promoter is indicated. Source data are provided as a Source Data file.

Article Snippet: ChIP and input DNAs were subjected to library preparation with NEBNext ChIP-seq Library Pre Reagent (NEB, E7370S).

Techniques: Infection, Labeling, ChIP-sequencing, RNA Sequencing Assay