title: H3K27ac HiChIP of iKAP metastatic CRC cell line, with Kdm5d deletion
design: HiChIP was performed following published protocol27. Briefly, the cells were fixed in 1% formaldehyde for 10 minutes at RT and quenched by glycine for 10 minutes. After washing with PBS, cells were resuspended in Hi-C lysis buffer and rotated at 4 C for 30 minutes. Nuclei were pelleted, washed with Hi-C Lysis Buffer and resuspended in 0.5% SDS, which was diluted and quenched by Triton X-100. NEB buffer 2 and Mbol restriction enzyme (New England Biolabs, Cat#R0147) were used to fragment the genome and the DNA fragments were labeled with biotin-dATP (Thermo Fisher). Proximal DNA fragments were then ligated using NEB T4 DNA ligase (New England Biolabs, Cat#M0202). The nuclear pellet was lysed in nuclear lysis buffer and chromatin was then sonicated using a Covaris E220 and clarified by centrifugation. Clarified samples were diluted in ChIP Dilution Buffer. Samples were precleared with Dynabeads Protein G (Invitrogen). H3K27ac antibody (Abcam, Cat#ab4729) were added at a concentration of 3.75 g per 5x106 cells and incubated overnight at 4 C. Dynabeads Protein G beads were used to capture the antibody-protein-DNA complex. The captured samples were washed with low-salt wash buffer, high-salt wash buffer and LiCl wash buffer. The DNA was reverse-crosslinked in DNA elution buffer and incubated with Proteinase K (Invitrogen). The DNA was purified using DNA Clean and Concentrator columns (Zymo Research) and eluted in water. After quantification using Qubit dsDNA HS Assay Kit (Thermo Fisher), Streptavidin C-1 beads (Thermo Fisher) were added to samples to capture the biotin-labeled DNA. After washed, DNA was mixed with Tn5 transposase (Illumina) for tagmentation for library preparation. DNA was amplified by Phusion HF (New England Biolabs), Nextera Ad1_noMx and Nextera Ad2.X (see reference for primer sequences). After removing the primers with Ampure XP beads (Beckman Coulter) cleanup, the Nextera_i7Short and Nextera_i5Short primers (see reference for primer sequences) were added to samples for additional PCR reactions. The DNA was size-selected using Ampure XP beads to capture fragments between 300bp and 700bp.