Chromatin immunoprecipitation sequencing (ChIP-seq)

ChIP-seq was performed from two biological replicates at four time points. At each time point, 3 × 10⁷ cells were harvested and fixed with 1% of formaldehyde for 8 min (Thermo Scientific, 28908). Formaldehyde was quenched with 0.2 M glycine for 5 min. Chromatin was sheared to 200–500 bp in size using the Covaris S220 ultrasonicator. A fraction of the resulting chromatin was used as input control. Sheared chromatin (50 μg for C/EBPα, 30 μg for H3K4me1, 100 μg for BRD4) was incubated with 5 μg of antibodies – anti-C/EBPα (Santa Cruz, sc-61), anti-H3K4me1 (abcam, ab8895), and anti-BRD4 (Bethyl Laboratories, A301–985A100) – bound to protein-A beads (Life Technologies, 9999–01). Samples were incubated at 65°C overnight to reverse the crosslinks, followed by RNase and proteinase K treatments. Purified ChIP and input DNA were quantified by Qubit 2.0 Fluorometer (Life Technologies, {"type":"entrez-protein","attrs":{"text":"Q32866","term_id":"75280873","term_text":"Q32866"}}Q32866). Libraries were generated using NEB Ultra DNA Library kit (NEB, E7370S) and amplified by nine cycles with KAPA Real-Time Library Amplification Kit (Peqlab, KK2701). The libraries of BRD4-ChIP-seq were sequenced at 2 × 75 bp on an Illumina NextSeq machine in house. The other libraries were sequenced at 2 × 50 bp on HiSeq2000 (TAL, Göttingen or LAFUGA, LMU München). The average read numbers obtained were 4 × 10⁷ for C/EBPα ChIP-seq, 7 × 10⁷ for H3K4me1 ChIP-seq, and 2 × 10⁷ for BRD4 ChIP-seq.