GSM1483872: KBM7-TM3C-1; Homo sapiens; OTHER
Approximately six million NHEK and ten million KBM7 cells were fixed in 1.5% formaldehyde at room temperature for 10 minutes. The fixed cells were washed with TN buffer (10 mM Tris, 40 mM NaCl, pH 7.5) and collected by centrifugation at 600 g for 3 minutes. To increase digestion efficiency, fixed cells (6 or 10 million / 122 ul) were treated with SDS (add 3.8 ul of 10% SDS to a final of 0.30% SDS) at 64°C for 10 minutes and then at 37°C overnight (15 hours). The SDS concentration was reduced gradually to 0.10% by adding five times of 50 ul (1 x DpnII digestion buffer or NEB buffer 4 for multiple enzymes) with mixing. Triton X-100 (38 ul of 20% Triton X) was added to 1.8% concentration and the sample was incubated at  37°C for 1 hour. Sample volume was adjusted to 600 ul by adding 1 X restriction buffer, ATP (0.2 mM final) and BSA (100 ug/ml final). Digestion with appropriate restriction enzymes (300 units each) was carried out on a rotate shaker at 37°C for 15 hours. We used high concentration NEB enzymes to keep the final volume of the enzyme mixture less than 60 ul (1/10 reaction volume). The digested samples were deactivated at 65°C for 15 minutes and then centrifuged at 15,000 g for 5 min. We recovered ~95% of cellular DNA in the pellet fraction. The pellet fraction was re-suspended with T4 ligation buffer (15 ul 10 x buffer, 65 ul total) heated at 65°C and mixed with 100 ul of melted 2.5% low-melting agarose. We used 200 ul pipette to deliver the hot agarose sample to ice-cold ligation buffer (800 ul of 1 x ligation buffer containing T4 ligase (4000 units, NEB) in a steady fashion within ~5 seconds, on melted ice. Strings of gel bead appeared instantly at 0°C. We sealed the tube with parafilm and perform ligation at RT (23°C.) overnight on top of a shaker (~300 rpm), then transfer the tube to a iced water bath. The sample pellet was recovered by centrifugation at 20,000 g for 2 minutes, then 10 ul of 1% SDS (0.05% final) was added and heated at 80°C for 1 hour. Cross-links were reversed by treatment with Proteinase K (200 ug/ml) at 65°C and 300 rpm overnight (12 hours). Melted TM3C-agarose sample was incubated with RNase A (10 ug / 210 ul) at 55°C for 15 minutes and then purified by QIAquick gel extraction protocol (QUIAGEN Inc., CA). Purified TM3C DNA was quantified using both a NanoDrop spectrophotometer (Thermo Scientific) and a Qubit 2.0 Fluorometer. The Qubit quantification represents the more accurate DNA concentration. Size-selected fragments were paired-end sequenced (100 bp per end) after adding sequencing adaptors using Illumina Hi-Seq 2000.
GEO Accession
GSM1483872