Sample information curated by ChIP-Atlas

Antigen

Antigen Class
Histone
Antigen
H3K27ac

Cell type

Cell type Class
Blood
Cell type
GM12873
Tissue
blood
Lineage
mesoderm
Description
B-lymphocyte, lymphoblastoid, International HapMap Project, CEPH/Utah pedigree 1459, treatment: Epstein-Barr Virus transformed

Attributes by original data submitter

Sample

source_name
B-lymphoblastoid cell line
cell line
GM12873
treatment
untreated
sample pairing
biological replicate: sample 15
antibody
H3K27ac

Sequenced DNA Library

library_strategy
ChIP-Seq
library_source
GENOMIC
library_selection
ChIP
library_construction_protocol
ChIP-Seq_trio: 20 million cells were crosslinked at RT, first with 2 mM di(N-succinimidyl) glutarate for 45 min and then with 1 % methanol-free formaldehyde for 10 min. Formaldehyde was quenched for 5 min at RT using 0.125 M glycine/PBS and cells were washed with ice-cold PBS. Nuclei were isolated by thoroughly resuspending cells in ChIP Lysis Buffer followed by high-speed centrifugation at 4°C (repeated 3 times) and sonicated using a probe-type sonicator in ChIP Lysis Buffer. Nuclear debris was sedimented using high-speed centrifugation at 4°C, the top 90% of the supernatant was diluted ten-fold with ChIP Lysis Buffer, then diluted chromatin equivalent to 5 million cells were immunoprecipitated overnight at 4°C with 2.5 μg anti-histone H3 (acetyl K27) antibody (Abcam, cat. ab4729) or isotype control antibody (Santa Cruz Biotechnology, cat. sc-2027 X). Immunoprecipitation reactions were centrifuged and the top 90% of the supernatant (antibody-antigen complexes) was captured for 6 hours at 4°C using 47.5 μl Protein A-Protein G paramagnetic bead mix (1:1 ratio) (Thermo Fisher Scientific, cat. 10002D and 10004D) pe-blocked overnight with 0.5 w/v% BSA/PBS at 4°C. Capture beads were washed for 3 min on a rotating rack at 4°C once with IP Wash Buffer I, twice with IP Wash Buffer II, once with IP Wash Buffer III and finally twice with IP Wash Buffer IV, collecting beads between washes using a magnetic rack (components of in-house buffers can be found in Table S3). Antibody-antigen complexes were eluted using 200 μl Bead Elution Buffer (30 min at RT, 1000 rpm), and crosslinks were reversed by adding 0.4 M NaCl and incubating overnight at 65°C. Eluates were then treated with 20 μg RNAse A and 40 μg Proteinase K, respectively. ChIP fragments were isolated using Quiagen's MinElute PCR purification kit (cat. 28006) as per the manufacturer's recommendations. ChIP-Seq (sGT and trio) Sequencing libraries were prepared following Illumina's TruSeq RNA Sample Preparation v2 Guide with poly(A) selection from 1 μg total RNA. Indexed libraries were pooled and sequenced on the NextSeq 500 platform (75 bp, single-end).

Sequencing Platform

instrument_model
NextSeq 500

hg38

Number of total reads
14520253
Reads aligned (%)
98.2
Duplicates removed (%)
7.8
Number of peaks
23184 (qval < 1E-05)

hg19

Number of total reads
14520253
Reads aligned (%)
97.8
Duplicates removed (%)
7.8
Number of peaks
23748 (qval < 1E-05)

Base call quality data from DBCLS SRA