Instrument: NextSeq 500
Strategy: ChIP-Seq
Source: GENOMIC
Selection: ChIP
Layout: PAIRED
Construction protocol: Fixed cells were lyzed on ice for 20 minutes (5 mM PIPES, 85 mM KCl, 0.5% Igepal-CA 630) before pelleting and nuclear lysis (50 mM Tris-HCl, 10 mM EDTA, 1% SDS), chromatin was then sonicated to 200bp in a Covaris S220 and incubated overnight with 2 µl of anti-H3K4me3 (07-473; Millipore) and Protein A and Protein G Dynabeads (Invitrogen). Beads were washed seven times with RIPA buffer variants (10 mM Tris-HCl, 1mM EDTA, 0.5mM EGTA, 1% Triton X-100, 0.1% SDS, 0.1% Sodium Deoxycholate): RIPA (twice), High Salt RIPA (500 mM NaCl; twice), RIPA with 250mM LiCl (once) and T.E. Buffer (twice) before treatment with RNAse A at 37ºC for 1 hr (Roche) and Proteinase K 65ºC overnight (Thermo Fisher). DNA was recovered by phenol-chloroform extraction. Libraries were indexed for sequencing using NebNext Ultra II (New England BioLabs); library profiles were visualized using D1000 tape on a TapeStation (Agilent Technologies) and quantified using a universal library quantification kit (KAPA Biosystems).