GSM8921789: AKTP MTO, RM-044, replicate 2; Mus musculus; Hi-C
For cell lysis, 600 μl lysis buffer (10 mM Tris-HCl Ph=8, 10 mM NaCl, 0.2% IGEPAL CA-630, 1x protease inhibitor complex (Roche, 11873580001) in water) was added to each pellet. Cells were incubated for 30 min on ice and immediately centrifuged for 5 min at 950 g at 4 ºC. All centrifugations throughout the protocol were done under the same conditions. Pellets were washed with 1x NEBuffer 2 (NEB, B7002S), resuspended in 190 μl 1x NEBuffer 2 and 10 μl 10% SDS and incubated for 10 min at 65 ºC 350 rpm. SDS effect was quenched with 400 μl 1x NEBuffer 2 and 120 μl 10% Triton X-100 (Merck, 648466) for 30 min at 37 ºC 300 rpm. Nuclei were spun down, washed with 1x NEBuffer 2 and resuspended in 300 μl 1x NEBuffer 2 with 400 Units of MboI (NEB, R0147M). Chromatin was digested overnight at 37 ºC 300 rpm. 1 ug of DNA was sheared in a Bioruptor Pico (Diagenode, B01080010) and pull-down of the biotin tagged Hi-C fragments was done through binding to Dynabeads MyOne Streptavidin T1 beads (ThermoFisher Scientific, 65601). Hi-C libraries were prepared using the NEBNext End Repair Module (E6050L), NEBNext dA-Tailing Module (E6053L) and NEBNext Quick Ligation Reaction Buffer 5X (B6058S). PCR amplification (8 cycles) was done with UDIs (E6440S) and NEBNext High-Fidelity 2x PCR Master Mix (M0541S). Amplified libraries were purified with 0.9x volume AMPure beads and eluted in water. Library concentration and size were assessed with Qubit HS dsDNA (Invitrogen, Q33321) and Tapestation (Agilent, 5067-5589, 5067-5590), respectively, and sequencing was done with Illumina NovaSeq 6000 2x150 bp paired-end sequencing for at least 1,000 M reads.