Illumina Genome Analyzer IIx sequencing; Oct4 ChIP-seq in Rex1GFPd2 mouse embryonic stem cell line
Oct4 ChIP-seq in Rex1GFPd2 mouse embryonic stem cell line
Rex1GFPd2 mouse ES cells were maintained in NDiff N2B27 media in the presence of CHIR99021 and PD0325901 as described in Yang et al., 2012. For time series of differentiation, the media containing inhibitor was removed and replaced with NDiff N2B27 media as time indicated. ES cells were cross-linked as described in Lee et al., 2006. Yang SH, Kalkan T, Morrisroe C, Smith A, Sharrocks AD. (2012) PLoS Genet. 2012;8(12):e1003112. A genome-wide RNAi screen reveals MAP kinase phosphatases as key ERK pathway regulators during embryonic stem cell differentiation. QIAGEN PCR extraction kit ChIP-seq was performed as described in Lee et al., 2006, with the following modifications. The crosslinked material was sheared by water bath sonication using biorupter, at high level, 30 secs on/ 30 secs off, 4  degrees C. Immunoprecipitation was performed by incubating 4x10^6 cells with 2 ug of Oct4 antibodies (N-19; Santa Cruz), overnight at 4 degrees C.  Lee TI, Johnstone SE, Young RA. (2006) Nat Protoc. 1(2):729-48. Chromatin immunoprecipitation and microarray-based analysis of protein location.
Experimental Factor: immunoprecipitate
Anti-Oct4
Experimental Factor: time
16 hour