Chromatin immunoprecipitation

Cross-linked chromatin from 3×10⁶ cells per sample was prepared with a truChIP low cell chromatin shearing kit with SDS shearing buffer according to the manufacturer's instructions (Covaris, Woburn, MA). Chromatin was sheared with a Covaris sonicator for 4 min according to the manufacturer's instructions. Sheared chromatin was immunoprecipitated with the MAGnify chromatin immunoprecipitation system according to the manufacturer's instructions (Life Technologies/Thermo Fisher Scientific). ChIP-grade antibodies anti-histone H3 (tri methyl K4) and anti-histone H3 (tri methyl K9) (Abcam) were used for immunoprecipitation. The concentration of the pulled-down chromatin was quantified with PicoGreen (Life Technologies/Thermo Fisher Scientific). Equal amounts of chromatin, 3 μl, were added to 100 μl of 1× PicoGreen and quantified on an Infinite M1000 plate reader (Tecan). The immunoprecipitated chromatin was then analyzed via quantitative PCR (qPCR), using SYBR green PCR master mix (Applied Biosystems, Warrington, UK) and MND-forward (5′-CCTGAAATGACCCTGTGCCTT-3′) and MND-reverse primers (5′-AGGGGTTGTGGGCTCTTTTAT-3′). The qPCR was run at the default settings on the ViiA 7 thermal cycler (Applied Biosystems).