Illumina Genome Analyzer IIx sequencing; FAIRE-seq in Rex1GFPd2 mouse embryonic stem cell line
FAIRE-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. Lee TI, Johnstone SE, Young RA. (2006) Nat Protoc. 1(2):729-48. Chromatin immunoprecipitation and microarray-based analysis of protein location. FAIRE-seq was performed as described in Simon et al., 2012, with the following modifications. The crosslinked material was sheared by water bath sonication using bio-rupter for 17 mins, at high level, 30 secs on/ 30 secs off, 4 C. The size of sheared DNA fragments was between 100 bp to 300 bp, with median size around 200 bp. Simon JM, Giresi PG, Davis IJ, Lieb JD. (2012) Nat Protoc. 7(2):256-67. Using formaldehyde-assisted isolation of regulatory elements (FAIRE) to isolate active regulatory DNA.
Experimental Factor: time
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