Illumina HiSeq 2000 sequencing; ChIP-Seq of OCT4, NANOG, p300 in 3iL Human Embryonic Stem Cells
Sample information curated by ChIP-Atlas
Antigen
Antigen Class
No description
Antigen
NA
Cell type
Cell type Class
Pluripotent stem cell
Cell type
hESC H1
NA
NA
Attributes by original data submitter
Sample
ENA first public
2013-12-05
ENA last update
2018-03-08
ENA-CHECKLIST
ERC000011
External Id
SAMEA2235099
INSDC center alias
GIS
INSDC center name
Genome Institute of Singapore
INSDC first public
2013-12-05T17:02:14Z
INSDC last update
2018-03-08T16:50:40Z
INSDC status
public
Submitter Id
E-MTAB-2044:hESCs
broker name
ArrayExpress
cell line
H1
cell type
embryonic stem cell
common name
human
sample name
E-MTAB-2044:hESCs
sex
male
Sequenced DNA Library
library_name
hESCs_Input
library_strategy
ChIP-Seq
library_source
GENOMIC
library_selection
ChIP
library_construction_protocol
The hESC lines H1 (WA-01) was used for this study. For routine culture of hESCs in TeSR1 (Stem cell technologies), cells were cultured feeder free on matri-gel (BD). Medium was changed daily. The hESCs were subcultured with 1 mg/ml Dispase (Stem cell technologies) every 5-7 days. For treatment with 3iL conditions, hESCs cultured in TeSR1 was treated with 3iL 48 hrs post-seeding. The cells are subsequently subcultured on mitomycin C inactivated mouse fibroblast. Cells are dissociated to single cells using TrypLE (Life technologies). 3iL medium was refreshed daily and cells were subcultured upon confluency. Crosslinking of protein and DNA was done at room temperature for 10 minutes using serum free media that contains 1% formaldehyde. The crosslinking reaction was quenched with 0.2M final concentration of glycine for 5 minutes. Cells were washed extensively with TBSE (20 mM Tris-HCl, 150 mM NaCl, 1 mM EDTA), 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 (50 mM HEPES-KOH, 150 mM NaCl, 2 mM EDTA, 1% Triton X-100, 0.1% deoxycholate, 0.1% SDS) and the DNA was sheared with the probe sonicator (Branson digital sonifier S-450D) for 20 cycles of 30 seconds with 1 min intervals at amplitude 40. The chromatin sample was centrifuged at 20,000 rpm for 45 mins before the supernatant was harvested as chromatin extracts for ChIP. Chromatin extracts were pre-cleared with Protein G Dynal Magnetic Beads (Invitrogen) (2 h, 4C) and immunoprecipitated overnight at 4C using Protein G Dynal Magnetic Beads pre-coupled with antibodies against Oct4 (Abcam ab19857), Nanog (R&D AF1997) or p300 (Santa Cruz sc585). Beads were washed 3x with 0.1% SDS buffer, once with 0.1% SDS/ 0.35M NaCl buffer, once in 10 mM Tris-HCl, 0.25M LiCl, 1 mM EDTA, 0.5% deoxycholate, 0.5% NP-40, and once in TE buffer (10 mM Tris-HCl, 1 mM EDTA). Immunoprecipitated material was eluted from the beads with 50 mM Tris-HCl, 10 mM EDTA, 1% SDS at 69C for 30 mins in a orbital shaker at 10000 rpm. Crosslinks were reversed by incubation with pronase for 2 h at 42C then 6 h at 67C. DNA was extracted by phenol/chloroform/isoamyl-alcohol followed by chloroform, then precipitated with ethanol and resuspended in TE buffer. ChIP-Seq library was prepared using the NEBNext ChIP-Seq Library kit (NEB Biolabs) according to the manufacturer’s instructions. Briefly, the DNA was end-repaired with exonucleases and an adaptor was ligated to the end. Size selection was performed using AMPure XP Beads prior to PCR amplification for 15-18 cycles. The amplified DNA was purified using AMPure XP Beads, multiplexed and sequenced with the HiSeq 2000 system (Illumina).