E-MTAB-1565:ERK2_Lane2
ChIP-Seq for ERK2/MAPK1 and ELK1 in human embryonic stem cells
ChIP-Seq for ERK2/MAPK1 and ELK1 in human embryonic stem cells
ERK2
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.
GIS
E-MTAB-1565:ERK2_Lane2
Experimental Factor: immunoprecipitate
ERK2