GSM4050755: LoVo ChIP-seq WDR5 rep2; Homo sapiens; ChIP-Seq
Sample information curated by ChIP-Atlas
TFs and others
Cell type Class
Attributes by original data submitter
Cell Signaling WDR5 (D9E1I)
Sequenced DNA Library
ChIP-seq assays (performed as described in Weissmiller, et. al. Nat Commun,10, Article number: 2014 (2019)): Briefly, cross-linking was carried out with 0.75% formaldehyde (Sigma-Aldrich; in PBS pH 7.4) at room temperature for 10 minutes. The reaction was quenched with 125 mM glycine for 10 minutes at room temperature, after which cells were washed twice with ice-cold PBS. Nuclei were prepared by incubating cells in Nuclear Lysis Buffer A (1 M HEPES pH 7.9, 1 M KCl, 0.5 M EDTA, 0.4% NP-40, PMSF, Na3VO4, and Complete Protease Inhibitor Cocktail) for 5 minutes on ice, after which they were collected by centrifugation. Nuclei were then lysed in Formaldehyde Lysis Buffer (50 mM HEPES pH 7.5, 140 mM NaCl, 1 mM EDTA, 1% Triton X-100, 1% SDS, PMSF, Na3VO4, and Complete Protease Inhibitor Cocktail) for 15 minutes on ice. Chromatin was sheared by 17–24 minute sonication (BioRuptor UCD-200, Diagenode; on highest setting, alternating between 30 seconds on and 30 seconds off) to yield a mean chromatin size of ~250 bp. After sonication, debris was cleared by centrifugation, and sheared chromatin diluted 10-fold in Formaldehyde Lysis Buffer without SDS. Chromatin from 107 cells was used per immunoprecipitation reaction. Immunoprecipitation reactions were performed with either 0.8 µL of IgG or 4.0 µL of α-WDR5 antibody as indicated. Immune complexes were recovered on Protein A agarose beads (Roche) and washed sequentially with Low Salt Wash buffer (20 mM Tris pH 8.0, 150 mM NaCl, 2 mM EDTA, 1% Triton X-100, PMSF, and Complete Protease Inhibitor Cocktail), High Salt Wash Buffer (20 mM Tris pH 8.0, 500 mM NaCl, 2 mM EDTA, 1% Triton X-100, PMSF, and Complete Protease Inhibitor Cocktail), LiCl Wash buffer (10 mM Tris pH 8.9, 250 mM LiCl, 1 mM EDTA, 1% Triton X-100, PMSF, and Complete Protease Inhibitor Cocktail) and TE (10 mM Tris pH 8.0, 1 mM EDTA, supplemented with PMSF and Complete Protease Inhibitor Cocktail). Protein–DNA complexes were de-crosslinked overnight at 65°C in 50 µL Elution Buffer (10 mM Tris pH 8.0, 1 mM EDTA, 200 mM NaCl, 0.1% SDS, and 20 µg Proteinase K (740506, Clontech). --- RNA-seq assays: CHP134 cells were lysed in Trizol (ThermoFisher), after which RNA was isolated using the Direct-zol RNA MiniPrep Kit (Zymo Research) with on-column DNase digestion, following the manufacturer's instructions. RNA-Seq analysis (with rRNA reduction) was performed at GENEWIZ. Library preparation with rRNA depletion and paired-end 150 base pair sequencing on an Illumina HiSeq was performed by GENEWIZ. --- SLAM-seq assays: Nascent RNA was labeled using the SLAMseq Kinetics Kit–Anabolic Kinetics Module (061, Lexogen). LoVo cells were pretreated with 25 uM C6, C6nc, or 0.1% DMSO prior to a three hour labeling with 1 mM 4-Thiouridine (S4U). Following the manufacturer's instructions, total RNA was isolated, alkylated with iodoacetamide, flash frozen, and shipped to Lexogen for analysis. For analysis of ChIP-Seq, de-crosslinked DNA samples from three ChIP reactions were pooled (a total of 3 x 107 cellular equivalents per sample), and DNA purified by phenol-chloroform extraction and recovered by ethanol precipitation. Indexed libraries were then made using the DNA Ultra II Library Prep Kit for Illumina (E7645, New England Biolabs). Library quality was assessed using the 2100 Bioanalyzer (Agilent) and libraries quantitated using KAPA Library Quantification Kits (KAPA Biosystems). Pooled libraries were subject to 75 bp single sequencing according to the manufacturer's protocol (Illumina NextSeq500). Sequencing was performed by the Vanderbilt Technologies for Advanced Genomics (VANTAGE) Shared Resource. Bcl2fastq2 Conversion Software (Illumina) was used to generate de-multiplexed Fastq files. --- RNA-Seq analysis (with rRNA reduction) was performed at GENEWIZ. Library preparation with rRNA depletion and paired-end 150 base pair sequencing on an Illumina HiSeq was performed by GENEWIZ. RNA-Seq for CHP-134 cells was completed with three biological replicates. --- For analysis of SLAM-seq: After quality control analyses, libraries were prepared (250 ng RNA per sample) using Lexogen's QuantSeq 3' mRNA-Seq Library Prep Kit FWD for Illumina, following the User Guide (015UG009V0251) recommendations. Sequencing was performed by Lexogen on an Illumina NextSeq 500 system, using the SR75 High Output Kit. The SLAMdunk analysis pipeline (34) was used to analyze SLAMseq sequencing data. SLAM-Seq completed with three biological replicates.