title: Illumina HiSeq 2500 paired end sequencing; XAB2 binds genome-wide on putative DNA elements (e.g. enhancers, promoters) that are known to regulate gene expression
design: XAB2 binds genome-wide on putative DNA elements (e.g. enhancers, promoters) that are known to regulate gene expression
library_construction_protocol: For ChIP assay,cells were cross-linked at room temperature for 2.5 min with 1% formaldehyde. Chromatin was prepared and sonicated on ice for 8 min using a Covaris S220 Focused-ultrasonicator. Samples were immunoprecipitated with antibodies (5-6μg) overnight at 4οC followed by incubation for 3 h with protein G Sepharose beads (Millipore) and washed sequentially. Naive mESCs were cultured on gelatinized tissue culture dishes in medium containing Dulbecco's modified Eagle's medium (DMEM) supplemented with 15% fetal bovine serum (FBS), 50μg mL-1 streptomycin, 50 U mL-1 penicillin (Gibco), 2mM L-glutamine (Gibco), 1% non-essential amino acids (Gibco), 0.1mM β-Mercaptoethanol (Applichem), 1μM MEK inhibitor PD0325901 (Selleck), 3μM GSK inhibitor CHIR99021 (Selleck). Differentiation was induced by substitution of LIF and inhibitors PD0325901/CHIR99021 with 10μM trans-retinoic acid (Sigma) for 7 days. The complexes were eluted and the crosslinking was heat reversed. Purified DNA fragments were analyzed by sequencing. Standard TruSeq ChIP library construction protocol