GSM8107899: HsSKM_LatB_Rep2; Homo sapiens; MNase-Seq
Control cells and Lat B treated cells were cross-linked with 1 mM dithiobis(succinimidyl propionate) (DSP) in PBS at room temperature for 30 min, quenched with 1 M Tris-HCl pH=8.0 at room temperature for 15 min and washed with PBS for 3 times. Fixed cells were lysed with 400 μl RIPA-G buffer + protease inhibitor cocktail (PIC ) +RNase to extract DNA-protein complex. Isolated DNA-protein complex was rinsed with 400 μl PBS 5 times and then suspended in 400 μl PBS. After centrifugation, the pellet was redissolved in 100 μl RIPA-T buffer +RNase by sonication (1s/1s, for 5s in total). The redissolved DNA-protein complex was digested with 0.5 μl Micrococcal Nuclease ( #10011,CST) at 37°C for 30 mins. After centrifugation, the DNA was loaded on 1.5% agarose gel, and then purified by E.Z.N.A. R Gel Extraction Kit (Omega_D2500-02; Omega BIO-TEK).  The DNA-protein complex absorbed on the column was incubated with digestion buffer (100 μl PBS + 2 μl Proteinase K biosharp#BS080-100mg)  to digest bound protein at 55°C for 2 hours. DNA was collected by centrifugation at 20,000 ✕ g for 1 min.  To de-crosslink DSP, 10 ul of 100 mM DTT was added into the eluate and incubated at 70℃ for 10 min. To remove proteinase K and DTT, the DNA sample was purified using E.Z.N.A. R Gel Extraction Kit. The library construction was carried out using TruSeq DNA PCR-free prep kit (20015963, illumina). Library was sequenced by NovaSeq Sequencer