Sample information curated by ChIP-Atlas

Antigen

Antigen Class
Histone
Antigen
H3K4me3

Cell type

Cell type Class
Pluripotent stem cell
Cell type
hESC H9
NA
NA

Attributes by original data submitter

Sample

source_name
Null_H3K4me3
cell line
H9
cell type
embryonic stem cell (ESC)
genotype/variation
EZH2 -/- (Null)
chip antibody
H3K4me3 (Abcam, ab8580)

Sequenced DNA Library

library_strategy
ChIP-Seq
library_source
GENOMIC
library_selection
ChIP
library_construction_protocol
Native ChIP for profiling histone marks was performed as described (Gilfillan et al., 2012) with minor modifications. Cell pellets (from 400,000 cells) were thawed and suspended in 95μl MNase Buffer (50mM Tris-HCl pH8, 1mM CaCl2, 0.2% Triton X-100) supplemented with 5mM Sodium Butyrate and 1X Complete EDTA-free Protease Inhibitor (Roche). Chromatin was digested with micrococcal nuclease (3U; NEB) for 8min at 37°C to obtain predominantly mononucleosomes and dinucleosomes. Stop buffer (10μl of 110mM Tris-HCl pH8, 5mM EDTA) was added to inactivate the reaction. Chromatin was solubilised by brief sonication using a Diagenode Bioruptor for 1min on high power. Chromatin was diluted in 100μl RIPA-IP buffer (280mM NaCl, 1.8% Triton X-100, 0.2% SDS, 0.2% Sodium Deoxycholate, 5mM EGTA supplemented with 5mM Sodium Butyrate and 1X Complete EDTA-free Protease Inhibitor) and insoluble material was removed by centrifugation at 14,000 rpm at 4°C for 15 minutes. Supernatant was transferred to a new tube, 10% was removed for the input sample, and the remaining chromatin was pre-cleared with 50μl pre-washed Protein A and Protein G Dynabeads (Life Technologies) at 4°C for 1 hour. Dynabeads were removed, the pre-cleared chromatin was diluted to 500μl in RIPA-IP buffer and split into 5 tubes. Chromatin was incubated overnight with histone antibodies at 4°C with rotation. Pre-washed Dynabeads (10μl) were added to each tube and incubated at 4°C for 2-3 hours with rotation. Beads were washed five times with RIPA buffer, once with LiCl buffer (250mM LiCl, 10mM Tris-HCl pH8, 0.5% NP-40, 0.5% Sodium Deoxycholate, 1mM EDTA) and once with 1xTE. Beads were suspended in 100μl 1X TE supplemented with 50μg Proteinase K and incubated at 55°C for 1 hour. ChIP and Input DNA were purified using Genomic DNA Clean and Concentrator columns (Zymo) and eluted in 50μl 1xTE. Crosslinked ChIP for profiling EZH2 occupancy was performed using iDeal ChIP-Seq kit for Transcription Factors (Diagenode). Indexed ChIP-Seq libraries were generated with the NEBNext Master Kit (NEB) using NEBNext Multiplex Oligos for Illumina indexes (NEB).

Sequencing Platform

instrument_model
Illumina HiSeq 2500

hg38

Number of total reads
21880986
Reads aligned (%)
96.5
Duplicates removed (%)
16.5
Number of peaks
26365 (qval < 1E-05)

hg19

Number of total reads
21880986
Reads aligned (%)
95.8
Duplicates removed (%)
17.7
Number of peaks
26287 (qval < 1E-05)

Base call quality data from DBCLS SRA