title: GSM7809991: hTSC, ST, day4, Hypoxia, H3K4me3, clone CT27, HiChIP, replicate1; Homo sapiens; OTHER
library_name: GSM7809991
library_construction_protocol: Approximately 10 million cells were fixed in 1% PFA for 10 min and 3 mM disuccinimidyl glutarate for 40 min as previously described. Fixed NIH3T3 cells were added to the cross-linked cells as spike-in controls. The cell mixtures were lysed in HiC Lysis Buffer [10 mM Tris-HCl (pH 7.5), 10 mM NaCl, and 0.2% IGEPAL CA-630], and the isolated nuclei were resuspended in 0.5% sodium dodecyl sulfate (SDS) and permeabilized at 62°C for 10 min. Next, a 3.3-fold volume of 1.5% Triton X-100 was added and incubated at 37°C for 15 min. In situ digestion with DpnII (NEB #R0543) was performed at 37°C for 60 min, and the enzyme was heat-inactivated at 62°C for 20 min. Overhangs were biotin-labeled using DNA Polymerase I large (Klenow) Fragment (NEB #M0210) at 37°C for 60 min in the presence of biotin-14-dATP (Thermo Fisher Scientific #19524016), dTTP, dCTP, and dGTP. Proximity ends were ligated with 13.3 units/µl of T4 DNA ligase (NEB #M0202) at 23°C for 4 h. The nuclei were pelleted and treated with 0.5 units/µL of Exonuclease III (NEB #M0206S) for 5 min at 37°C, resuspended in Nuclear Lysis Buffer [50 mM Tris (pH7.5), 10 mM EDTA, and 1% SDS], and sonicated using Covaris M220 (M&S Instruments Inc.) to 300-700 bp length. Sonicated DNA was diluted 1:10 with ChIP Dilution Buffer [50 mM Tris (pH 7.5), 165 mM NaCl, 1.1% Triton X-100, and 0.01% SDS] and clarified by centrifugation. An anti-H3K4me3 antibody (Merck Millipore, Clone #CMA304) was added to the fragmented DNA at 1:500 dilution, and the mixture was rotated at 4 °C for 60 min. The IP complex was captured using Dynabeads M280 anti-mouse (Life technologies #11201D), and the beads were washed four times with High-Salt Wash Buffer [20 mM Tris (pH 7.5), 500 mM NaCl, 2 mM EDTA, 1% Triton X-100, 0.1% SDS, and 0.1% sodium deoxycholate] and twice with 10 mM Tris-HCl (pH7.5). The washed beads were suspended in Extraction Buffer [10 mM Tris (pH8.0), 350 mM NaCl, 0.1 mM EDTA, and 1% SDS] supplemented with 5% volume of proteinase K (Takara, #9034), and incubated at 55°C for 1 h and 67°C for 2 h to reverse the crosslinks. ChIPed DNA was purified using MinElute PCR Purification Kit (QIAGEN #28004). For biotin pull-down, 5 µl of Dynabeads MyOne Streptavidin C1 beads (Life Technologies #65001) were suspended in 2x Biotin Binding Buffer [10 mM Tris-HCl (pH 7.5), 1 mM EDTA, and 2 M NaCl] and mixed with an equal volume of ChIPed DNA. After 50 min of rotation at room temperature, the beads were washed with Tween Wash Buffer [5 mM Tris-HCl (pH 7.5), 0.5 mM EDTA, 1 M NaCl, 0.05% Tween-20] and NEB T4 Ligase Buffer (NEB # B0202), and resuspended in 10 mM Tris-HCl (pH 8.0). HiChIP libraries were constructed using KAPA Hyper Prep Kit (NIPPON Genetics #7962312001) and TruSeq-compatible duplex Y adapter (IDT). After PCR amplification, the amplicon was size-selected using Ampure XP (Beckman Coulter #A63880).