ChIP-seq for Tbx3 in Mus musculus hepatocellular carcinoma
HCC (H1 and H4), and ICC (E9 and E10) cell lines were grown in DMEM high glucose glutamax (GIBCO) media in 600 cm2 tissue culture dishes to 80% confluence. 50 million cells were harvested in 10 million cell aliquots in 15 mL media. Each aliquot was cross-linked in 1% paraformaldehyde for 10 minutes at room temperature. The cross-linking was quenched with the addition of 1mL of 2M glycine, and incubated at room temperature for 5 minutes. Chromatin was isolated and digested with 1.2  L of Micrococcal Nuclease using the Cell Signalling SimpleChIP kit (#9002). 40 million cell equivalents of pre-cleared chromatin was used to perform immunoprecipitation following the Cell signalling protocol. Immunoprecipitations for Tbx3 with HCC - H1 and HCC - H4 chromatin were performed with AB sc-17871 (5  g), and the IP for Prdm5 with ICC - E9 and ICC - E10 chromatin were performed with AB MABE972 (5 g). Inputs were derived from 500 000 cell equivalents of chromatin. ChIP-seq libraries were produced using a modified protocol from the Accel-NGS 2S Plus DNA Library Kit (#21024), where we performed DNA extraction using 25:24:1 phenol:chloroform:isoamyl alcohol followed by overnight ethanol precipitation of DNA at each step of the protocol. Additionally, we followed an enrichment for small DNA fragments outlined in the X-ChIP protocol(10.7554/eLife.09225). ChIP-seq libraries underwent quality control using the Agilent Technologies 4200 Tapestation (G2991-90001) and quantified using the Invitrogen Qbit DS DNA HS Assay kit (Q32854). Libraries were sequenced in single-end 65bp on an Illumina HiSeq 2500, generating 262M reads (33M in average per sample).