GSM7850110: MCF7_MED1KR_HiCAR; Homo sapiens; OTHER
About 400,000 post-confluent cells were digested and fixed with 1% formaldehyde for 10 min, followed by quenching with 125mM glycine and washed with cold TBS (20mM Tris-HCl, 150mM NaCl, pH 7.4) once. Cells were lysed by NPB buffer (PBS with 5% BSA, 0.2% NP-40, 1mM DTT) and nuclei were spinned down and re-suspended with TB buffer (33mM Tris-AC, 66mM KAc, 10mM MgAC2, 16% DMF, pH 7.8) where tagmentation by assembled Tn5 (Diagenode) complex was performed. The nuclei were further lysed by 0.5% SDS at 62°C and 1.25% Triton X-100 at 37°C and then digested by CviQI (New England) followed by Tn5 adapter ligation which required help from splint oligo in RCB buffer (50mM Tris-HCl, 50mM NaCl, 0.2% SDS, pH 8.0). Next, cross-links were reversed and DNA was precipitated in glycogen-NaAC-Ethanol solution and re-suspended in Tris-HCl (10mM, pH 8.0) where Nla III digestion was performed. AMPure XP beads (Beckman Coulter) was used for size selection and DNA at 1 ng/μl was circularized by T4 DNA ligase (New England) in Tris-HCl (10mM, pH 8.0). Final library was amplified by Q5 polymerase (New England). The PCR condition: initial denaturation 98°C for 30 sec, 12 cycles of denaturation 98°C for 10 sec/ annealing 59°C for 30 sec/ extension 72°C for 30 sec, then final extension 72°C for 5 min. The size of library should range from 200bp to 800bp, so the DNA with smaller or larger size was removed by another round of AMPure XP beads purification. The sequencing was performed with Illumina NovaSeq SP (100bp two ends).