title: ChIP-seq for Prdm5 in Mus musculus intrahepatic cholangiocarcinoma
design: 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).