Comment[ArrayExpressAccession]	E-MTAB-2042
MAGE-TAB Version	1.1
Investigation Title	ChIP-Seq of STAT3 in 3iL Human Embryonic Stem Cells
Comment[Submitted Name]	ChIP-Seq of STAT3 in 3iL Human Embryonic Stem Cells
Experiment Description	Human embryonic stem cells (hESCs) are isolated from the inner cell mass of the blastocysts.  The pluripotent properties of hESCs enable the derivation of cell-types or tissues of different lineages for potential applications such as therapeutics discovery and regenerative medicine.  Even though hESCs are pluripotent, differences have been observed when compared to the native pluripotent epiblast cells of the blastocyst. We use a chemical approach (3iL: 3 small molecule inhibitor and cytokine) to induce an expression signature that more closely resembles native pluripotent cells. This experiment is STAT3 binding data in 3iL hESCs.
Experimental Design	cell_type_comparison_design	in_vitro_design	ChiP-seq
Comment[AEExperimentType]	ChIP-seq
Experimental Factor Name	cell type	immunoprecipitate
Experimental Factor Type	cell type	immunoprecipitate
Quality Control Type
Quality Control Term Source REF	EFO
Public Release Date	2013-12-05
Person Last Name	Goke	Chan	Ng
Person First Name	Jonathan	Winston Yun Shen 	Huck-Hui
Person Mid Initials
Person Email	gokej@gis.a-star.edu.sg	chanysw@gis.a-star.edu.sg	nghh@gis.a-star.edu.sg
Person Phone
Person Address	60 Biopolis Street, Genome, #02-01, Singapore 138672	60 Biopolis Street, Genome, #02-01, Singapore 138672	60 Biopolis Street, Genome, #02-01, Singapore 138672
Person Affiliation	Genome Institute of Singapore	Genome Institute of Singapore	Genome Institute of Singapore
Person Roles	submitter;investigator	investigator	investigator
PubMed ID
Publication Author List	Yun-Shen Chan, Jonathan Goke, Jia-hui Ng, Xinyi Lu, Cheng-Peow Tan, Wei-Quan Tng, Zhong-Zhi Hong, Huck-Hui Ng
Publication Title	A LIF-dependent human pluripotent state with a distinctive regulatory circuitry
Publication Status	submitted
Protocol Name	P-MTAB-35802	P-MTAB-35803	P-MTAB-35804	P-MTAB-35805	P-MTAB-35806
Protocol Type	growth protocol	treatment protocol	nucleic acid extraction protocol	nucleic acid library construction protocol	nucleic acid sequencing protocol
Protocol Description	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.	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 Phospho-Stat3 (Cell Signalling Technology 9145). 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).	Samples were multiplexed and sequenced as single reads of length 51bp on the HiSeq 2000 Illumina platform.
Protocol Term Source REF	EFO	EFO	EFO	EFO	EFO
Protocol Hardware					Illumina HiSeq 2000
Term Source Name	EFO	ArrayExpress
Term Source File	http://www.ebi.ac.uk/efo	http://www.ebi.ac.uk/arrayexpress
Comment[SecondaryAccession]	ERP004237
Comment[SequenceDataURI]	http://www.ebi.ac.uk/ena/data/view/ERR361904
SDRF File	E-MTAB-2042.sdrf.txt