title: GSM6588206: MIDHiC_WT_mESC_R5; Mus musculus; OTHER
library_construction_protocol: mESC cells were double crosslinked using formaldehyde (Fisher Scientific, 50-980-494) and DSG (ProteoChem, c1104-100mg) and aliquoted as described previously (Hsieh et al., 2020). One aliquot of fixed cells (~5 million) was washed in 1.4 mL low SDS buffer (50 mM pH 7.5 Tris-HCL, 10 mM NaCl, 0.1% SDS) with 1X Protease Inhibitor Cocktails (PICs; Roche, 11873580001). Incubate on ice for 15 min. Spin down the nuclei at 2500 rcf for 5 min at 4C. Remove supernatant and wash one more time in the low SDS buffer on ice. Then, resuspend nuclei in 1 mL digestion buffer (1X Cutsmart buffer, 1% Triton X-100, 1X PICs). Incubate at 37C for 20 min with gentle rotation. Add 250U MseI (NEB, R0525M) and 250U DdeI (NEB, R0175L) to digest nuclei at 37C for 6 hours or overnight with gentle rotation. After digestion, spin down the nuclei and resuspend in 1 mL fill-in reaction mix (1X CutSmart buffer, 1% Triton X-100, 66 uM dTTP, 66 uM dCTP, 66 uM dGTP, 66 uM dATP-Biotin (Jena Bioscience, JBS-NU-835-BIO14-L), 50 U Klenow DNA large polymerase I (NEB, M0210M), 1X PICs). Rotate at room temp for 90 min. Spin down nuclei and resuspend in 1 mL ligation mix (1X T4 DNA ligase buffer, 1% Triton X-100, 0.1 mg/mL BSA, 15 U T4 ligase (Life Technologies, 15224041), 1X PICs). Incubate at 14C overnight with gentle rotation. Next day, spin down the nuclei. The proximal ligated nuclei can be used for library preparation or proceeded to MID HiChIP. Resuspend the proximal-ligated nuclei from MID Hi-C 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 Phenol:Chloroform:Isoamyl (PCI) method. 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 8-10 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.