title: GSM6588216: MIDHiChIP_H3K27ac_WT_mESC_R1; Mus musculus; OTHER
library_construction_protocol: Proximal-ligated nuclei from MID Hi-C experiment were processed for ChIP following Myers Lab ChIP-seq Protocol (v011014). In brief, proximal-ligated nuclei were resuspended in RIPA buffer followed by sonication using Bioruptor. Soluble chromatin fragments are immunoprecipitated by SMC3 (abcam ab9263) or H3K27ac (ActiveMotif 39133) antibody overnight. We used 3 to 5 mg of antibody for the starting material of 5 million cells. Next day, wash the immunoprecipitated chromatin extensively. Purify the DNA for library preparation after reverse crosslinking. Resuspend the proximal-ligated nuclei from MID HiChIP experiment in 300 uL ChIP elution buffer (1% SDS, 100 mM NaHCO3) and incubate at 65C overnight. Next day, add RnaseA and incubate at 37C for 45 min. Then, add Proteinase K and incubate at 55C for 2 hours. Purify DNA using ChIP DNA Clean & Concentrator kit from Zymo (D5205). Purified DNA can be stored in -80C till ready for library preparation. Before generating sequencing library, prepare Dynabeads MyOne Streptavidin T1 (Invitrogen, 65601) beads by washing 5 uL T1 beads twice in 600 ul Tween wash buffer (1X binding buffer, 0.05% Tween-20) at 55 C for 2 min. Reclaim the beads on magnetic stand and resuspend in 50 ul 2X binding buffer (2 M NaCl, 10 mM pH 7.5 Tris-HCl, 1 mM EDTA). The beads are ready for immobilizing biotin-labeled DNA. We used NEB fragmentase (NEB, M0348S) to fragment DNA. But sonication can be used to fulfill the same purpose. 50 ng to 500 ng DNA were treated with NEB fragmentase in 20 uL reaction volume, per manufacture instructed. Incubate the reaction at 37C for 9 to 13 min, optimized to generate 100 – 500bp fragment size. To stop the reaction, add 250 ul 2X binding buffer to the DNA fragmentation reaction and incubate at 55C for 15 min. Then, add 280 uL Zymo DNA elution buffer (Zymo Research, D3004-4-50) and 50 ul prepared T1 beads to the stopped reaction (total 600 ul). Rotate the beads and DNA mix at room temperature for 15 min. After that, reclaim the beads and wash twice in the Tween wash buffer as described above. The DNA-bound T1 beads were used for NEBNext Ultra II DNA Library Preparation (NEB, E7645S) per manufacture instruction with gentle taps during reactions to keep beads in suspension. We typically amplify 10-15 PCR cycles for library amplification. The library was then double-size selected using home-made SPRI beads to isolate fragments between 200 and 800 bp for next generation sequencing.