Illumina HiSeq 2500 paired end sequencing; ChIP-sequencing of hepatocyte-like cells (HLCs) and primary human hepatocytes (PHHs)
ChIP-sequencing of hepatocyte-like cells (HLCs) and primary human hepatocytes (PHHs)
Chromatin was crosslinked with 1% formaldehyde for 10 minutes at RT and quenched with 0.125M glycine. Cells and nuclei were lysed and chromatin was sonicated to fragment DNA to about 200-500 bp on a Bioruptor Pico sonication device (Diagenode). Sonicated chromatin was pre-cleared with same-host IgG and protein G Dynabeads. For immunoprecipitation, 100µg of chromatin was incubated with 2µg of each antibody: H3K27ac (Abcam, ab4729), H3K4me1 (Abcam, ab8895),  H3K27me3 (Active Motif, 39155) and H3K4me3 (Merk, 05-745R) overnight at 4C and complexes captured with 30µl of protein G Dynabeads. Complexes were washed, RNAse and Proteinase K treated. DNA was purified by phenol-chloroform extraction. A sonicated chromatin sample (1%) was also collected as input for normalisation. One input (input 1) was collected for H3K27ac+H3K4me1 immunoprecipitation and a second input (input 2) for H3K27me3+H3K4me3.  For hIPSC and hESC derived hepatocyte-like cells (HLCs), 2 independent differentiation experiments were used. Cells were differentiated into HLCs for 30 days has previously reported (Hannan et al, 2013) with minor modifications. Following 3 days of endoderm differentiation, anterior foregut specification was achieved with RPMI-B27 differentiation media supplemented with 50ng/ml Activin A (R&D) for 5 days. Cells at the foregut stage were further differentiated into hepatocytes with Hepatozyme complete medium: HepatoZYME-SFM (Thermo Fisher) supplemented with 2mM L-glutamine (Thermo Fisher), 1% penicillin-streptomycin (Thermo Fisher), 2% non-essential aminoacids (Thermo Fisher), 2% chemically defined lipids (Thermo Fisher), 14μg/ml of insulin (Roche), 30μg/ml of transferrin (Roche), 50 ng/ml hepatocyte growth factor (R&D), and 20 ng/ml oncostatin M (R&D), for up to day 30 of differentiation. Adult primary human hepatocytes (PHHs) were purchased from Biopredic International as a monolayer culture. One biological replicate was used and immunoprecipitated as 2 separate technical replicates.  Immunoprecipitated DNA was purified by phenol-chloroform extraction and precipitated with GlycoBlue (Thermo Fisher), sodium acetate (Thermo Fisher) and ethanol (Sigma-Aldrich).  Library preparation was performed by the Wellcome Trust Sanger Institute DNA Sequencing Facility (Hinxton, UK). ChIP-sequencing libraries were generated using 10ng of DNA and 8 PCR cycles for all samples.