title: H3K4me3 HiChIP of immortalized human aortic endothelial cells treated with TNFα against untreated controls
design: H3K4me3 HiChIP of immortalized human aortic endothelial cells treated with TNFα against untreated controls
library_name: TeloHAEC_H3K4me3_control_rep2_p
library_construction_protocol: TeloHAECs were grown in cell culture. 5 million TeloHAECs per replicate were crosslinked in 37% formaldehyde as per instructions of the Arima Hi-C kit (cat# A510008) and frozen down as 106 cells per aliquot. After DNA estimation, cells corresponding to 3 ug of total DNA were thawed on ice and lysed in 20 ul of cold lysis buffer for 15 min, followed by permeabilization, digestion, and proximity ligation as per the instructions of the Arima Hi-C kit (cat# A510008).We skipped step 10 of the Enzyme D step in the Arima kit and proceeded to ChIP. For ChIP, we  adapted the protocol from Fang et al. 2016 with slight modifications. After proximity ligation, the nuclei were spun down at 2,500 g for 5 min at 4°C and the supernatant was discarded. The nuclei were then resuspended in 130 μl RIPA buffer (10 mM Tris, pH 8.0, 140 mM NaCl, 1 mM EDTA, 1% Triton X-100, 0.1% SDS, 0.1% sodium deoxycholate) with proteinase inhibitors. The nuclei were lysed on ice for 10 min and then sonicated using Diagenode Bioruptor® Pico with the following setting: 10 Cycles of 30 Secs On/Off. After sonication, 370 ul of RIPA buffer was added to the samples before they were cleared by centrifugation at 20,000 G for 20 min and the supernatant was collected. The clear cell lysate was mixed with 500ul of antibody (2.5 μg of H3K4me3, Millipore cat #04-745) coated Dynabeads M-280 (Sheep anti-Rabbit Thermo Scientific cat #11201D) and rotated at 4°C overnight. Next day, the beads were collected by centrifugation at 2,000 rpm for 1 min and then resuspended in 1 ml of RIPA buffer and washed for three times in 1x RIPA buffer, twice in high-salt RIPA buffer (10 mM Tris, pH 8.0, 300 mM NaCl, 1 mM EDTA, 1% Triton X-100, 0.1% SDS, 0.1% sodium deoxycholate), once with LiCl buffer (10 mM Tris, pH 8.0, 150 mM LiCl, 1 mM EDTA, 0.5% IGEPAL CA-630, 0.1% sodium deoxycholate), and twice with TE buffer (10 mM Tris, pH 8.0, 0.1 mM EDTA). Washed beads were first treated with 10 μg Rnase A in extraction buffer (10 mM Tris, pH 8.0, 350 mM NaCl, 0.1 mM EDTA, 1% SDS) by shaking at 1000 rpm for 1 h at 37°C. Then 20 μg proteinase K was added and reverse crosslinking was performed by shaking at 1000 rpm overnight at 65°C or at least 2 h. The fragmented DNA was purified with DNA Concentrator (Zymo Research, cat# D4014). Lastly, the biotin pull-down procedure was performed according to Swift Biosciences Accel-NGS 2S Plus DNA Library Kit (cat# 21024) An optimal number of PCR cycles were determined by qPCR using the KAPA library quantification kit (cat# KK4824). Libraries were checked on a bioanalyzer for quality and then sequenced