Sample information curated by ChIP-Atlas

Antigen

Antigen Class
Histone
Antigen
H3K36me3

Cell type

Cell type Class
Pluripotent stem cell
Cell type
ES cells
NA
NA

Attributes by original data submitter

Sample

source_name
Embryonic stem cells
cell type
Embryonic stem cells
genotype
WT
passage
p60
antibody
H3K36me3

Sequenced DNA Library

library_strategy
ChIP-Seq
library_source
GENOMIC
library_selection
ChIP
library_construction_protocol
For ssDRIP-seq, 5 x 106 cells were harvested, lysed by SDS (final concentration: 0.5%) and treated with proteinase K (NEB, final concentration: 0.1 mg/ml) at 37 ºC overnight. Genomic DNA was extracted using the classic phenol-chloroform method. The genomic DNA was fragmented by endonucleases (Mse I, Dde I, Alu I and Mbo I; NEB) at 37 ºC overnight. Then, DRIP was performed as previously described (Xu et al., 2017). For ChIP-seq, ChIP assay was performed as reported in previous studies with some minor modifications (Dahl and Collas, 2008). Briefly, 1 x 106 cells were harvested and crosslinked using 1% formaldehyde at room temperature for 6 min and then quenched with 2.5 mol/L glycine at room temperature for 5 min. The cells were then lysed in lysis buffer (1% SDS, 50 mM Tris-HCl, 10 mM EDTA) for 10 min and sonicated to 250-bp fragments using M220 Focused-ultrasonicator (Covaris, USA). The samples were incubated overnight with protein A beads (Thermo Fisher Scientific) linked with 2.4 µg antibody. After reverse-crosslinking and elution at 68 ºC, the IPed DNA was extracted by the aforementioned phenol-chloroform method. For RNA-seq, total RNA was extracted using TRIzol (Thermo Fisher Scientific) from 1 x 106 cells per experimental replicate. For ATAC-seq, 3 x 104 cells were lysed in the lysis buffer (10 mM Tris-HCl, pH 7.4, 10 mM NaCl, 3mM MgCl2, 0.5% NP-40) for 10 min on ice. Then spun at 500 g for 5 min to remove the supernatant, followed by adding 50 μL transposition reaction mix from TruePrepTM DNA Library Prep Kit V2 for Illumina (Vazyme Biotech) at 37 ºC for 30 min. For WGBS, genomic DNA was extracted using DNeasy Blood & Tissue Kits (Qiagen) according to the manufacturer's instructions. For ssDRIP-seq, libraries were constructed using ACCEL-NGS 1S plus DNA Library Kit (Swift, USA) following manufacturer's instructions. For ATAC-seq, library amplified and purified using TruePrep DNA Library Prep Kit V2 for Illumina(Vazyme Biotech). For ChIP-seq, the enriched fragments were constructed into libraries without the incorporation of spike-in controls via KAPA Hyper Prep Kits with PCR Library Amplification/Illumina series (KK8504) following the manufacturer's instructions. For RNA-seq, sequencing libraries were generated using NEBNext® Ultra™ RNA Library Prep Kit for Illumina® (NEB, USA) following manufacturer's recommendations. For WGBS, genomic DNA were sonicated to 200 - 300 bp fragments followed by end repair and adenylation. Cytosine-methylated barcodes were added before bisulfite treatment. After the quantification of DNA concentration by Qubit® 2.0 FlurometerFluorometer (Life Technologies, CA, USA) and quantitative PCR, the libraries were sequenced on an Illumina Hiseq platform.

Sequencing Platform

instrument_model
HiSeq X Ten

hg19

Number of total reads
70135965
Reads aligned (%)
68.1
Duplicates removed (%)
13.2
Number of peaks
1751 (qval < 1E-05)

hg38

Number of total reads
70135965
Reads aligned (%)
68.5
Duplicates removed (%)
13.1
Number of peaks
2158 (qval < 1E-05)

Base call quality data from DBCLS SRA