title: GSM6046938: Lenti-X 293T,FAIRE-rep1; Homo sapiens; FAIRE-seq
library_construction_protocol: Briefly, cells were fixed with 1% formaldehyde for 10 minutes, and the residual formaldehyde was quenched with 0.125M glycine. The cell pellet from each 10cm cell culture dish was collected and washed once with cold 1× PBS buffer and lyzed in 1 ml Hypotonic lysis buffer containing Protease Inhibitor (MCE, Cat#HY-K0010). The nuclei were pelleted by a 12000rpm centrifuge at 4°C and washed once with cold 1x PBS buffer. The pellet was then resuspended in 0.1% SDS lysis buffer and incubated for 60 minutes at 4°C. Chromatin was sonicated to a fragment size of 200-300bp using Bioruptor (Bioruptor pico). The chromatin lysate was cleared by centrifugation at 12000 rpm for 5 minutes at 4°C. For each FAIRE reaction, 100 μl chromatin lysate was supplied with 400 μl 1× TE buffer (pH = 8.0) and subjected to twice phenol/chloroform/isoamyl alcohol (25:24:1) extraction, and then chloroform/isoamyl alcohol extraction. The DNA-containing water layer was transferred to a new 1.5 ml tube and supplied with 1/10 volume of 3M sodium acetate, two volumes of 100% ethanol, and 1 μl of 20 mg/ml glycogen (Thermo Fisher Scientific, Cat# R0551). After incubating at -80°C for 2 h, the DNA was precipitated by centrifugation and washed once with 70% ethanol. Subsequently, the DNA was resuspended in 10 mM Tris-HCl (pH=7.4) and reverse cross-linked through overnight incubation at 64°C in the presence of Proteinase K and 0.3M NaCl. Thereafter, RNaseA (Thermo Fisher Scientific) was supplied to remove the trace amount of RNA with incubation at 37°C for 30 min. The DNA was purified with VAHTS DNA Clean Beads (Vazyme, Cat#N411-01) and subjected to both qPCR assay and FAIRE-seq analysis. 100 ng FAIRE DNA/Input DNA was employed for library preparation using NEBNext® Ultra™ DNA Library Prep Kit for Illumina