Sample information curated by ChIP-Atlas

Antigen

Antigen Class
TFs and others
Antigen
GATA4

Cell type

Cell type Class
Pluripotent stem cell
Cell type
hESC derived endodermal cells
NA
NA

Attributes by original data submitter

Sample

source_name
dEN_shGATA4_3
cell line
HUES64
cell type
dEN shGATA4
chip antibody
GATA4(R&D Systems,AF2606,VAZ0312041)

Sequenced DNA Library

library_strategy
ChIP-Seq
library_source
GENOMIC
library_selection
ChIP
library_construction_protocol
Cells were grown to a final count of 10 million, resuspended in PBS, and crosslinked in 10% formaldehyde solution for 10 minutes at room temperature. Following quenching with 0.125M glycine and two PBS washes, we isolated nuclei using cell lysis buffer (20 mM Tris-HCl ph8, 85mM KCl, 0.5% NP40). Nuclei were then digested using MNase (Worthington, LS004797) as done in (Henikoff et al., PNAS 2011). Digestion was stopped with 0.05M EGTA and chromatin was aliquoted into 1-2 million cells per ChIP. Antibodies were added and immunoprecipitation was carried out overnight at 4°C as done in1. The next day, protein G beads (Life Technology, 10009D) were added for 2 hours at 4°C to isolate the protein bound DNA and washed twice using Low Salt Wash Buffer (0.1% SDS, 1% Triton X-100, 2mM EDTA, 20mM Tris-HCl pH 8.1, 150mM NaCl), High Salt Wash Buffer (0.1% SDS, 1% Triton X-100, 2mM EDTA, 20mM Tris-HCl pH 8.1, 500mM NaCl), LiCl Wash Buffer (0.25M LiCl, 0.5% NP40, 0.5% sodium deoxycholate, 1mM EDTA, 10mM Tris-HCl pH 8.1,), and TE Buffer pH 8 (10mM Tris-HCl, pH 8, 1mM EDTA pH 8). DNA was eluted twice using 100µL of ChIP Elution Buffer (1% SDS, 0.1M NaHCO3) at 65°C for 15 minutes. Crosslinking was reversed by addition of 32µl reverse crosslinking salt mixture (250 mM Tris-HCl pH 6.5, 62.5 mM EDTA pH 8, 1.25 M NaCl, 5mg/ml Proteinase K) for 5-18 hours at 65°C. DNA was isolated using phenol/chloroform extraction and treated with DNase-free RNase for 30 minutes at 37°C. The whole cell extract (WCE) control was generated using MNase treated material that was then reverse crosslinked and phenol chloroform extracted, skipping the immunoprecipitation and washing steps. MNChIP-seq library construction was performed as in (Henikoff et al., PNAS 2011) with gel size selection from 140bp to 720bp as described in (Gu et al., 2010). RRBS was performed according to a previously published protocol (Smith et al., 2009) with some optimizations for small cell numbers (Gu et al., 2010).

Sequencing Platform

instrument_model
Illumina HiSeq 2000

hg19

Number of total reads
15974400
Reads aligned (%)
165.2
Duplicates removed (%)
16.1
Number of peaks
10172 (qval < 1E-05)

hg38

Number of total reads
17779580
Reads aligned (%)
166.0
Duplicates removed (%)
15.5
Number of peaks
11037 (qval < 1E-05)

Base call quality data from DBCLS SRA