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Sample GSM3495181 Query DataSets for GSM3495181
Status Public on Aug 08, 2019
Title KO_4H8_Rep1_S3
Sample type SRA
Source name KO_4H8_mESCs
Organism Mus musculus
Characteristics strain background: 129
genotype/variation: TDG KO
cell type: mESCs
chip antibody: anti-RNA pol II (Millipore, #05-623)
Growth protocol Tdg knockout mouse embryonic stem cells (Tdg KO mESC) and wildtype control mESC were a gift from Dr. Primo Schar. Complete mESC media was composed of KnockOut DMEM (Gibco), supplemented with 2 mM GlutaMAX (Gibco), non-essential amino acids (Gibco), 0.1 mM 2-mercaptoethanol (SIGMA), 1,000 U/ml LIF (Millipore, #ESG1106), and 15% ES-tested FBS (Gibco, #10439-024). mESC were co-cultured with mitotically-arrested mouse embryonic fibroblast (MEF; produced in-house or purchased from Millipore, #PMEF-CF) on gelatin-coated (Millipore, #ES-006-B) dishes. MEF depletion was achieved by three 10-minute rounds of serial-plating onto uncoated tissue culture dishes. Human CD4+ human T lymphocytes were acquired and cultured as described in Marina et al., 2016.
Extracted molecule genomic DNA
Extraction protocol Prior to chromatin immunoprecipitation, antibodies (5 μl polyclonal or 5 μg monoclonal) were pre-bound to 200 μl Protein G magnetic beads (Invitrogen, #10004D) by overnight incubation in PBS with 5% BSA; beads were washed and resuspended in PBS with 5% BSA. MEF-depleted mESC were fixed at room temperature with 1% formaldehyde (Sigma-Aldrich, # 252549) for 5 min; fixation was stopped by adding glycine (ICN Biomedical, #ICN808822) to 125 mM. Cell membranes were lysed with cold NP-40 buffer (1% NP40, 150 mM NaCl, 50 mM Tris–HCl; pH 8.0) and nuclei collected by centrifugation at 12,000 × g for 1 min at 4 °C. Nuclear pellets were resuspended at a concentration of 2E8 cells/ml in ChIP sonication buffer (1% SDS, 10 mM EDTA, 50 mM Tris–HCl; pH 8.0) supplemented with protease (Thermo Scientific, #78430) and phosphatase (Calbiochem, #524627) inhibitor cocktails. Chromatin was sheared into 150-400 bp fragments by sonication (Bioruptor Twin, Diagenode). Debris was pelleted by centrifugation and cleared chromatin was diluted 10-fold in ChIP dilution buffer (1.1% Triton X-100, 0.01% SDS, 167 mM NaCl, 1.2 mM EDTA, 16.7 mM Tris–HCl; pH 8.1). The prepared antibody-bound beads were added to 1 ml diluted chromatin containing 2E7 million cell equivalents and incubated overnight with rotation at 4 °C. Immune complexes were washed 5 times with LiCl wash buffer (250 mM LiCl, 1% NP-40, 1% sodium deoxycholate, 100 mM Tris-HCl; pH 7.5) and once with TE (0.1 mM EDTA, 10 mM Tris-HCl; 7.5). Beads were resuspended in IP Elution Buffer (1% SDS, 0.1 M NaHCO3) and crosslinks reversed by overnight incubation at 65 °C. DNA was purified by column purification (QIAGEN, # 28106)
Sequencing libraries were constructed from DNA samples with the Illumina TruSeq V3 library construction protocol.
Library strategy ChIP-Seq
Library source genomic
Library selection ChIP
Instrument model NextSeq 550
Data processing ChIP-seq reads were aligned to mm10 using Bowtie version 2.2.9 with default parameters. Depth normalized read densities were produced with deepTools.
Peak calling was performed with MACS (v 2.2.1), using sonicated chromatin (input) as the control
For RNA-Seq, reads were trimmed, and aligned using Tophat 2.1.1. Gene expression values were computed with Cufflinks v.2.2.1 using Ensembl rel.83 annotation for mm10
Genome_build: mm10
Supplementary_files_format_and_content: bigWig files include depth normalized read density tracks
Supplementary_files_format_and_content: bed files include peak calling results; text (Cuffdiff output)
Submission date Nov 29, 2018
Last update date Aug 08, 2019
Contact name David Sturgill
Phone 240-760-6725
Organization name NIH
Department NCI
Street address Building 41, Rm B622
City Bethesda
State/province MARYLAND
ZIP/Postal code 20892
Country USA
Platform ID GPL21626
Series (1)
GSE123101 TET-catalyzed 5-carboxylcytosine promotes CTCF binding to suboptimal sequences genome-wide
BioSample SAMN10497182
SRA SRX5077385

Supplementary file Size Download File type/resource 187.5 Mb (ftp)(http) BW
SRA Run SelectorHelp
Raw data are available in SRA
Processed data provided as supplementary file

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