∼200-300 hPSC-derived endocrine progenitors clumps were dissociated using TrypLE Express Enzyme cross-linked with 3.3 mg/ml of dimethyl 3,3'-dithiobispropionimidate and 1 mg/ml of 3,3'-dithiodipropionic acid di(Nhydroxysuccinimide ester) (both Sigma Aldrich) for 30 minutes at room temperature and with 1% formaldehyde (Amresco) for 15 minutes. The cross-linking reaction was quenched with 0.125 M glycine and cells were first lysed in cell lysis buffer (10 mM Tris-HCl pH 8, 10 mM NaCl and 0.2% NP-40) and then in nuclear lysis buffer (50 mM Tris-HCl pH 8, 10 mM EDTA and 1% SDS) on ice in the presence of protease inhibitors on ice. Nuclear lysates were diluted in IP dilution buffer (20 mM Tris-HCl pH 8, 2 mM EDTA, 150 mM NaCl, 0.01% SDS and 1% Triton X-100) and sonicated for 30s on/45s off for 12 cycles using a Q500 sonicator (QSonica) with microtip probes at 30% power. Sonicated samples were pre-cleared using 10 μg rabbit IgG (Santa Cruz) and Protein A/G agarose beads. Agarose beads were removed by centrifugation and a portion of the supernatant was collected as the input control. Samples were divided equally and incubated with 10 μg of HNF1A antibody (Abcam ab96777) or rabbit IgG overnight at 4°C. The following day, samples were incubated with Protein A/G agarose beads and the beads were recovered and washed with IP wash buffer and Tris-EDTA buffer. The immunoprecipitated DNA was eluted from the beads using IP elution buffer (100 mM NaHCO3, 1% SDS, 100 mM DTT). Samples were successively treated with RNaseA, NaCl and Proteinase K. DNA was extracted by phenol/chloroform extraction. For ChIP-seq, libraries were prepared using the Solexa ChIP-seq DNA Sample Prep Kit (IP102-1001). After amplification, DNA fragments between 180 and 400 bp were gel extracted and sent for sequencing. Libraries were sequenced by the Genome Institute of Singapore using the Illumina Genome Analyzer II as single-end 35 bp reads
GEO Accession
GSM4147117