GSM8336675: 9464D_KO_MH2A1_REP2; Mus musculus; OTHER
CUT&RUN reactions were performed as described in Meers et al., 2019 (PMID: 31232687) following the “Standard CUT&RUN” protocol. Briefly, freshly harvested 1M 9464D cells were bound to concanavalin-A paramagnetic beads (Epicypher), then split equally, resuspended in antibody binding buffer and incubated overnight with either home-made macroH2A1 antibodies (described in Buschbeck et al., 2009 PMID: 19734898. and Douet et al., 2017 PMID: 28283545) and an IgG non-targeting control (Abcam ab46540). Both antibodies were diluted 1:100 in the binding reaction. Samples were then washed and bound with pA/G-MNase (Epicypher), chromatin digestion started by addition of CaCl2 and stopped after 30min with STOP buffer containing chelating agents. Samples were then incubated for 30min at 37oC to release CUT&RUN fragments, incubated 1h at 50oC with proteinase K and purified using ChIP DNA Clean & Concentrator (Zymo Research). Sequencing libraries were prepared with the KAPA HyperPrep kit (Roche) and NEXTflex DNA barcodes for Illumina (Bioo Scientific)