GSM6998277: Cerebellum, RREB1-3V5 ChIP, rep2; Mus musculus; ChIP-Seq
Cerebella from two non-pooled P21 B6J.Tg(PCP2-V7-3V5) mice were frozen in liquid nitrogen. Tissue was minced in 1% formaldehyde-PBS and incubated 10 minutes, rotating, at room temperature. Glycine was added at a final concentration of 0.125M to terminate crosslinking and samples were incubated an additional 10 minutes and then washed three times in PBS. After the final wash, tissue was resuspended in 600µL ChIP lysis buffer (50mM Tris-HCl pH 7.5, 0.150M NaCl, 1mM EDTA, 1% Igepal CA-630(Sigma, I8896), 0.25% Sodium deoxycholate, 0.10% SDS, protease inhibitor cocktail (Roche, 11836170001)), homogenized with a plastic pestle and incubated on ice 10 minutes.  Chromatin immunoprecipitation was performed as previously described (Texari et al. 2021). Briefly, lysate was sonicated for 10 seconds on, 30 seconds off, for 12 cycles at 20% amplitude with 2 minutes rest on ice, then 10 cycles at 30% amplitude with 2 minutes rest on ice, followed by 12 cycles at 30% amplitude. A microliter of each sample was run on the Tape Station using high sensitivity D1000 screen tape (Agilent, 5067- 5584) to test sonication efficacy. A 5µL aliquot of each sample was set aside as input and 50µL of V5 antibody conjugated to magnetic beads (MBL International, M167-11) was added to the remaining lysate and incubated overnight at 4˚C. After 3 washes with 200µL wash buffer I (20mM Tris-HCl pH 7.5, 150mM NaCl, 0.10% SDS, 1% Triton X-100, 2mM EDTA, 1x protease inhibitors) and 3 washes with 200µL wash buffer II (250mM LiCl, 1% Triton-X 100, 0.70% sodium deoxycholate, 10mM Tris-HCl pH 7.5, 1mM EDTA, 1x protease inhibitors), and 2 washes in 180µL TET buffer (10mM Tris-HCl pH 7.5, 1mM EDTA, 0.20% Tween 20, 1x protease inhibitors), bead-bound samples were resuspended in 80µL elution buffer (10mM Tris pH 8.0, 0.50% SDS, 5mM EDTA, 280mM NaCl) and reverse crosslinked with 1µL proteinase K (20mg/mL) for 1 hr at 55˚C followed by 1 hr at 65˚C. Library preparation was performed on beads as previously described (Texari et al., 2021) using the NEB DNA Ultra II Kit (NEB, E7645S). Adapters used were from KAPA (KAPA, KR1317).