title: GSM7728061: HepG2_siControl_IgG_rep2; Homo sapiens; OTHER
library_construction_protocol: CUT&RUN reactions were performed as described in Meers et al., 2019 following the “Standard CUT&RUN” protocol. Briefly, approximately 1M cryopreserved HepG2 cells were thawed and 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 and Douet et al., 2017) 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 CaCl 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).