GSM4673321: SpenKO#C3(iXist-ChrX)_3hDox_H2AK119ub1; Mus musculus; ChIP-Seq
Native ChIP-seq using buffers supplemented with 10 mM of the deubiquitinase inhibitor N-ethylmaleimide (Sigma, E3876-5G) throughout. Briefly, 50 million ES cells (or for 24h data sets, 40 million mES cells with 10 million Drosophila SG4 cells as spike-in) were lysed in RSB (10 mM Tris pH 8, 10 mM NaCl, 3 mM MgCl2, 0.1% NP40) for 5 min on ice with gentle inversion. Nuclei were resuspended in 1 ml of RSB + 0.25 M sucrose + 3 mM CaCl2, treated with 200 U of MNase (Fermentas) for 5 min at 37 °C, quenched with 4 µl of 1 M EDTA, then centrifuged at 5000 r.p.m. for 5 min. The supernatant was transferred to a fresh tube as fraction S1. The chromatin pellet was incubated for 1 h in 300 µl of nucleosome release buffer (10 mM Tris pH 7.5, 10 mM NaCl, 0.2 mM EDTA), carefully passed five times through a 27G needle and then centrifuged at 5000 r.p.m. for 5 min. The supernatant from this S2 fraction was combined with S1 as soluble chromatin extract. For each ChIP reaction, 100 µl of chromatin was diluted in Native ChIP incubation buffer (10 mM Tris pH 7.5, 70 mM NaCl, 2 mM MgCl2, 2 mM EDTA, 0.1% Triton) to 1 ml and incubated with 4ul H2AK119ub1 Ab (Cell Signalling Technology, #8240) overnight at 4 °C. Samples were incubated for 1 h with 40 µl protein A agarose beads pre-blocked in Native ChIP incubation buffer with 1 mg/ml BSA and 1 mg/ml yeast tRNA, then washed for a total of four times in Native ChIP wash buffer (20 mM Tris pH 7.5, 2 mM EDTA, 125 mM NaCl, 0.1% Triton) and once in TE. The DNA was eluted with 1% SDS and 100 mM NaHCO3, and was purified using the ChIP DNA Clean and Concentrator kit (Zymo Research). Libraries were prepared from 50ng ChIP DNA using NEBNext Ultra II DNA Library Prep Kit
GEO Accession
GSM4673321