GSM8718536: HME1_MSL3ko_H4K16acAb2_Rep1; Homo sapiens; ChIP-Seq
2x106 cells per pull down were cross-linked using 1% formaldehyde for 10 min. The reaction was stopped by incubation for 5 min with 0.125M glycine. After nuclear extraction and chromatin shearing by sonication, lysates were incubated overnight at 4°C with protein G Dynabeads (Thermo Fisher Scientific, 10003D) coupled with one of the following antibodies: anti-H4K16ac (Millipore, 07-329, 5g), anti-H4K16ac (Cell Signaling Technology, 13534; 1g) anti-H3K36me3 (Cell Signaling Technology, 4909S, 5l). After immunoprecipitation, beads were recovered using a magnet and washed; chromatin was eluted and cross-links reverted overnight at 65°C. DNA was purified with solid-phase reversible immobilization (SPRI) beads (Agencourt AMPure XP, Beckman Coulter), and then quantified with QuantiFluor (Promega, E2670). For H4K16ac pull-downs, Drosophila spike-in was added to normalize signal between Ctrl-KO and MSL3-KO cells. S2 Drosophila cells were processed as described above and lysates were quantified using DC Protein Assay Kit I (Bio-Rad Laboratories Ltd, 5000111). 5% chromatin was added to HME1 lysates and pull-down was carried on. DNA libraries were prepared using the NEBNext UltraII DNA Library Prep Kit for Illumina (New England Biolabs, E7645L).