Sample information curated by ChIP-Atlas

Antigen

Antigen Class
TFs and others
Antigen
MAPK1

Cell type

Cell type Class
Pluripotent stem cell
Cell type
hESC H1
NA
NA

Attributes by original data submitter

Sample

Alias
E-MTAB-1565:ERK2
Broker name
ArrayExpress
Description
Protocols: H1 hESCs were maintained as feeder-free culture on matri-gel (BD). The medium containing 20% KO serum replacement, 1 mM L-glutamine, 1% non-essential amino acids, 0.1 mM 2-mercaptoethanol and 4ng/ml basic fibroblast growth factor (Invitrogen) in DMEM/F12 (GIBCO) was conditioned with mitotically inactivated mouse embryonic fibroblast (MEF) for 24hrs. Additional 8ng/ml of basic fibroblast growth factor (Invitrogen) was supplemented to conditioned medium before use. Medium was changed daily. The hESCs were passaged with 1 mg/ml collagenase IV (Gibco) upon confluency The ERK2 antibody used for the ChIP is from Santa Cruz (D-2) and ELK1 antibody used for the ChIP is from Santa Cruz (I-20). Between 10e7-10e8 cells were used for the ChIP experiment. Crosslinking of protein and DNA was done at room temperature for 10 minutes with serum free media that contains 1% formaldehyde. The crosslinking reaction is quenched with 0.2M final concentration of glycine for 5 minutes. Cells were washed extensively with 1XPBS, scraped and pelleted. The cells were lysed with 0.1% SDS buffer and collected nuclei were lysed with 1% SDS buffer. Pelleted chromatin was resuspended in the 0.1% SDS buffer (10 mM Tris-HCl [pH 7.5], 150 mM NaCl, 0.1% SDS, 1 mM EDTA) and the DNA was sheared via sonication with the BioRuptor (Diagenode) for 16 cycles of 30 seconds with 1 min intervals at amplitude 40. The chromatin sample was precleared at 20,000rpm for 45mins before the eluents are used for ChIP. ChIP was performed with 5ug of the antibody using Magnetic Protein A beads (Invitrogen). The chromatin was incubated with the immobilized antibody overnight. The ChIP sample was washed 3 times with the IP (0.1%SDS) buffer, once with 1M NaCl in the same buffer, once with LiCl buffer and once with TE-buffer and DNA-protein complexes were eluted with 50 mM Tris-Cl (pH 7.5), 10 mM EDTA, 1% SDS at 69C for 30mins in a orbital shaker at 14000 rpm. The protein-DNA complex was dissociated with 1.5ug/ml Pronase at 42C for 2 hr followed by 67C for 6 hr. ChIP DNA from 2 duplicates were pooled for 10ng of starting material for the library construction. The ChIP enriched DNA was modified with the ChIP-Seq DNA Sample Prep Kit (IP-102-1001, Illumina). Briefly, the DNA end was repaired with exonucleases and an adaptor was ligated to the end, followed by PCR amplification for 15 rounds. The amplified DNA was gel purified and the 200-300bp fragments were selected for subsequent SOLEXA sequencing (Illumina) using the Illumina 1G instrument according to the manufacturers protocol for 36 bp reads.
INSDC center alias
GIS
INSDC center name
Genome Institute of Singapore
INSDC first public
2013-06-03T13:10:15Z
INSDC last update
2018-03-08T16:19:53Z
INSDC status
public
SRA accession
ERS226580
Sample Name
ERS226580
Title
ERK2
cell line
H1_hESC
cell type
human embryonic stem cells
sex
male

Sequenced DNA Library

library_name
ERK2
library_strategy
ChIP-Seq
library_source
GENOMIC
library_selection
ChIP
library_construction_protocol
H1 hESCs were maintained as feeder-free culture on matri-gel (BD). The medium containing 20% KO serum replacement, 1 mM L-glutamine, 1% non-essential amino acids, 0.1 mM 2-mercaptoethanol and 4ng/ml basic fibroblast growth factor (Invitrogen) in DMEM/F12 (GIBCO) was conditioned with mitotically inactivated mouse embryonic fibroblast (MEF) for 24hrs. Additional 8ng/ml of basic fibroblast growth factor (Invitrogen) was supplemented to conditioned medium before use. Medium was changed daily. The hESCs were passaged with 1 mg/ml collagenase IV (Gibco) upon confluency The ERK2 antibody used for the ChIP is from Santa Cruz (D-2) and ELK1 antibody used for the ChIP is from Santa Cruz (I-20). Between 10e7-10e8 cells were used for the ChIP experiment. Crosslinking of protein and DNA was done at room temperature for 10 minutes with serum free media that contains 1% formaldehyde. The crosslinking reaction is quenched with 0.2M final concentration of glycine for 5 minutes. Cells were washed extensively with 1XPBS, scraped and pelleted. The cells were lysed with 0.1% SDS buffer and collected nuclei were lysed with 1% SDS buffer. Pelleted chromatin was resuspended in the 0.1% SDS buffer (10 mM Tris-HCl [pH 7.5], 150 mM NaCl, 0.1% SDS, 1 mM EDTA) and the DNA was sheared via sonication with the BioRuptor (Diagenode) for 16 cycles of 30 seconds with 1 min intervals at amplitude 40. The chromatin sample was precleared at 20,000rpm for 45mins before the eluents are used for ChIP. ChIP was performed with 5ug of the antibody using Magnetic Protein A beads (Invitrogen). The chromatin was incubated with the immobilized antibody overnight. The ChIP sample was washed 3 times with the IP (0.1%SDS) buffer, once with 1M NaCl in the same buffer, once with LiCl buffer and once with TE-buffer and DNA-protein complexes were eluted with 50 mM Tris-Cl (pH 7.5), 10 mM EDTA, 1% SDS at 69C for 30mins in a orbital shaker at 14000 rpm. The protein-DNA complex was dissociated with 1.5ug/ml Pronase at 42C for 2 hr followed by 67C for 6 hr. ChIP DNA from 2 duplicates were pooled for 10ng of starting material for the library construction. The ChIP enriched DNA was modified with the ChIP-Seq DNA Sample Prep Kit (IP-102-1001, Illumina). Briefly, the DNA end was repaired with exonucleases and an adaptor was ligated to the end, followed by PCR amplification for 15 rounds. The amplified DNA was gel purified and the 200-300bp fragments were selected for subsequent SOLEXA sequencing (Illumina) using the Illumina 1G instrument according to the manufacturers protocol for 36 bp reads.

Sequencing Platform

instrument_model
Illumina Genome Analyzer II

hg19

Number of total reads
25024600
Reads aligned (%)
90.8
Duplicates removed (%)
4.3
Number of peaks
6803 (qval < 1E-05)

hg38

Number of total reads
25024600
Reads aligned (%)
92.9
Duplicates removed (%)
3.2
Number of peaks
6971 (qval < 1E-05)

Base call quality data from DBCLS SRA