GSM7462195: ZNF274KO_H3K9_WCE_3; Homo sapiens; ChIP-Seq
ChIP was performed as described previously (Imbeault et al., 2017). Briefly, cells were cross-linked for 10 minutes at room temperature in PBS + 1% methanol-free formaldehyde solution, followed by quenching with 250 mM Tris pH 8. Nuclei were sonicated with Covaris E220 sonicatior (settings: 5% duty, 200 cycle, 140 PIP, 20 min) to yield genomic DNA fragments with a bulk size 200-500bp. IP was performed overnight at 4º C using 5 μg antibody (anti-H3K9me3 Diagenode, ref. C15410056; anti-HA.11 BioLegend ref: 901503; anti-H3K4me3 Cell Signaling, ref. 9751S; anti-CTCF Millipore, ref. 07-729; anti-Rad21 Abcam, ref. ab993) coupled to 50µl Protein G Dynabeads (Invitrogen ref: 10009D). Final DNA was purified with QIAGEN Elute Column. Up to 10 ng of material for both total inputs and chromatin immunoprecipitated samples were used for library preparation. After end-repair and A-tailing, Illumina IDT indexes were ligated to the samples. Libraries were size-selected using Ampure XP beads (Beckman Coulter), quality-checked on a Bioanalyzer DNA high sensitivity chip (Agilent) and quantified with a Qubit dsDNA HS assay.  CuT&Tag was performed as described previously (Kaya-Okur et al., 2019). For each mark, 200'000 cells were used per sample using the anti-H3K9me3 primary antibody (Active Motif, AB_2532132). A homemade purified ProtA-Tn5 protein (3XFlag-pA-Tn5-Fl, addgene #124601) was produced and coupled with MEDS oligos by the Protein Production and Purification of EPFL, as previously described (Bryson T, Henikoff S. 3XFlag-PATn5 Protein Purification and MEDS-Loading; 2019. doi:10.17504/protocols.io.8yrhxv6). Purified recombinant protein was used at a final concentration of 700 ng/uL. Libraries were sequenced with 75 bp paired end on the NextSeq 500 (Illumina). NextSeq v2.5 (with Illumina protocol #15048776)