title: GSM5840324: hif1a_hyp_d2_br2; Mus musculus; ChIP-Seq
library_construction_protocol: Chromatin extracts were prepared as described in (Brookes et al., 2012). Briefly, cells were fixed with 1% formaldehyde (Thermo, 28906) in PBS at RT for 10 minutes. Then, 0.125M glycine (Sigma, 50046) was added to quench formaldehyde at RT for 5 minutes. Cells were washed twice with ice-cold PBS. To lyse, fixed cells were treated with swelling buffer (25mM HEPES pH 7.9, 1.5mM MgCl2, 10mM KCl (Invitrogen, AM9640G), 0.1% Igepal 630, 1x protease inhibitor cocktail, 1mM PMSF, 2mM NaVO3, 5mM NaF) at 4°C for 10 minutes. Cells were scraped on ice and passed thought 18G needles before centrifugation at 3000g, 4°C for 5 minutes. Cell pellet, corresponding to nuclei was carefully resuspended in sonication buffer (50mM HEPES pH 7.9, 140mM NaCl, 1mM EDTA, 1% Triton X-100, 0.1% Sodium-deoxycholade (Thermo, 89904), 0.1% SDS (Invitrogen, AM9822), 1mM PMSF, 2mM NaVO3, 5mM NaF) and incubated on ice for 10 minutes before sonication. Chromatin was sheared to an average size of 200-300bp with E220 Evolution Covaris sonicator for 6 cycles, 1 minute each. Shearing efficiency was checked by agarose gel Library preparation was performed using KAPA Hyper Prep Kit (Kapa Biosystems, KR0961) (input amount 10ng DNA, adapter concentration 1.5 µM and size selection of 200-700-bp after PCR with cycle number = 15) and following manufacturer's instructions.