H3K27Ac and H3K4me1 ChIP-seq in Rex1GFPd2 mouse embryonic stem cell line
H3K27Ac and H3K4me1 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 lindicated. 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. Lee TI, Johnstone SE, Young RA. (2006) Nat Protoc. 1(2):729-48. Chromatin immunoprecipitation and microarray-based analysis of protein location. 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 bio-rupter for 45 mins, at high level, 30 secs on/ 30 secs off, 4 ⁰C. The size of sheared DNA fragments was between 100 bp to 600 bp, with median size around 300 bp. Immunoprecipitation was performed by incubating 0.75×107cells with 10 µg of H3K27Ac and H3K4me1 antibodies (Abcam; ab8895 and ab4729), respectively, cross-linked to dynabeads protein A (Invitrogen), overnight at 4 ⁰C. 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-H3K27Ac
Experimental Factor: time
0 hour