GSM3263200: HCC1599_untreated_SMC1a_HiChIP; Homo sapiens; OTHER
HiChIP was performed as described (Mumbach et al., 2016) using antibody against Smc1 (Bethyl, cat# A300-055A). Briefly, 2 x 107 cells were crosslinked with 1% formaldehyde (Thermo Scientific, cat# 28908) for 10 min and subsequently quenched with 0.125M glycine (Invitrogen, cat# 15527-013). Chromatin was digested using MboI restriction enzyme (NEB, Cat#R0147), followed by biotin incorporation with Biotin-14-dATP (Invitrogen, 19524-016) in end-repair step, ligation, and sonication. Sheared chromatin was 4-fold diluted with ChIP dilution buffer (16.7mM Tris pH 7.5, 167mM NaCl, 1.2mM EDTA, 0.01% SDS, 1.1% Triton X-100) and cleared and then incubated with anti-Smc1 antibody at 4°C for overnight. Chromatin-antibody complexes were captured by Protein-A magnetic beads (Pierce, cat# 88846) and subsequently washed with Low Salt Wash Buffer, High Salt Wash Buffer, LiCl Wash Buffer and eluted. DNA was purified with MinElute PCR Purification Kit (Qiagen, cat# 28004) and quantified using Qubit dsDNA HS Assay Kit (Invitrogen, cat# Q32851). 50-150ng was used for capture with Dynabeads MyOne Streptavidin C-1 (Invitrogen, cat# 65001) and an appropriate amount of Tn5 enzyme was added to captured DNA to generate sequencing library. Paired-end sequencing (38 bp+38 bp) was performed on a NextSeq 550.
GEO Accession
GSM3263200