NextSeq 500 sequencing; SMAD2/3 chromatin immunoprecipitation sequencing (ChIP-seq) in human naïve and primed human pluripotent stem cells
SMAD2/3 chromatin immunoprecipitation sequencing (ChIP-seq) in human naïve and primed human pluripotent stem cells
hPSCs were washed once with PBS and crosslinked straight into the plates using HEPES buffer containing 1% formaldehyde at RT, 10mM Dimethyl 3,3′-dithiopropionimidate dihydrochloride (DTBP, Sigma) and 2.5mM 3,3′-Dithiodipropionic acid di(N-hydroxysuccinimide ester) (DSP, Sigma). Chromatin immunoprecipitation (ChIP) was performed as previously described (Brown et al., 2011). 10µg of SMAD2/3 antibody per ChIP was used (AF3797), and samples were purified using the iPure v2 bead kit (Diagenode). Libraries were constructed using the MicroPlex Library Preparation Kit v2 (Diagenode) following the manufacturer's instructions, 10ng of input and all of the ChIP DNA was used as the starting material. Libraries were quantified using KAPA Library Quantification Kit (Roche) following the manufacturer's instructions.