GSM6615286: H3.1_NC_I_HA; Mus musculus; ChIP-Seq
ChIP was carried out as detailed in Gutin et all, Cell reports 2019, with following modifications. Crosslinked pellet was re-suspended in 20ul of cell lysis buffer (50mM Tris pH8.0, 150mM NaCl, 1%TritonX-100, 0.1%sodium deoxycholate, 5 mM CaCl2, EDTA-free protease inhibitor cocktail and incubated on ice for 15 minutes. Cells were centrifuged at 3000RPM for 10 minutes at 4°C. The supernatant was discarded, and pellet was re-suspended in 10ul of lysis buffer supplemented with 17.5 units of micrococcal nuclease (Worthington MNase, LS004798) and chilled on ice for 10 minutes. Samples were incubated at 37°C for 15 minutes. To stop reaction, 20mM EDTA was added and samples were vortexed and placed on ice for 30 minutes. Cell lysate was then centrifuged at max speed (20,000xg) for 10 minutes at 4°C and supernatant moved to a separate tube. Reaction volume was increased to 180ul with RIPA buffer (10mM Tris pH8.0, 140mM NaCl, 1mM EDTA, 0.1%SDS, 0.1% sodium deoxycholate, 1% Triton X-100, EDTA-free protease inhibitor cocktail), and reaction was then split into two separate wells of 96-well LoBind Eppendorf plate. ~5ug of anti-HA (12CA5) and anti-myc (9E10) antibodies was added to the separate wells and incubated at 4°C for 2 hours with rotations. Both antibody stocks were the supernatant of the respective hybridoma cell cultures grown in miniPERM bioreactors in-house by the Weizmann Institute Core Facility Antibody Unit. All subsequent steps were carried out as described in (Gutin et al., 2018). SLIM-Chip, as per Gutin et al, Cell Reports 2019