GSM9230021: A549-sg; Homo sapiens; OTHER
4C-seq was performed as previously described using Hind III for the first digestion, and Dpn II for the second digestion. In brief, cells were cross-linked in 2% formaldehyde for 10 min at room temperature and quenched with 0.2M glycine. Cells were then rinsed for 2 times with ice-cold PBS at room temperature and harvested and incubated in lysis buffer (50 mM Tris-Cl, 150 mM NaCl, 5 mM EDTA, 0.5% CA630, 0.5% Triton X-100 and protease inhibitor cocktail) while tumbling for 1 h at 4 °C. Cells were then pelleted, washed, and resuspended in Hind III buffer. SDS was added to a concentration of 0.3% for 1 h in a thermomixer at 37 °C and 400 rpm. Triton X-100 was then added to a concentration of 1.8% and cells were incubated in a thermomixer at 37 °C at 400 rpm for 3 h. 200 units of Hind III enzyme were added for a 4 h incubation in a thermomixer at 37 °C and 900 rpm; another 200 units of Hind III were added for overnight incubation. Cells were then incubated 20 min at 65 °C to heat inactivate enzyme, suspended in ligation buffer and 4,000 units of T4 ligase and incubated overnight at 16 °C. The next day, 300 μg Proteinase K was added for overnight incubation at 65 °C. Ligated DNA was purified and digested with 90 units Dpn II in Dpn II buffer overnight in a thermomixer at 37 °C and 500 rpm, followed by heat inactivation of enzyme (65 °C for 25 min). Samples were then diluted with ligation buffer and 100 units ligase were then added for overnight incubation at 16 °C. Circularized DNA was PCR-amplified using bait-specific inverse primers. Finally, PCR products were pooled and purified. Libraries were prepared according to Illumina's instructions accompanying the Paired-End DNA Samples Prep Kit. Briefly, DNA fragment end repair, add A base to 3'end, and connect sequencing adapter. PCR amplified with Illumina primers for 15 cycles and library fragments of ~250 bp were band isolated from an agarose gel. The purified DNA was captured on an Illumina flow cell for cluster generation. Libraries were sequenced on an Illumina sequencer following the manufacturer's protocols.