title: GSM6133299: H9 cells, INDUCE-seq, rep 1; Homo sapiens; OTHER
library_construction_protocol: Cells were harvested using Accutase to obtain a single cell suspension and counted, followed by two washes in DPBS. ~ 100,000 cells were deposited into a well of a Poly-D-lysine coated 96 well plate. Once sedimented, cells were crosslinked in 4% PFA for 10 min followed by two washes with PBS. Plates were tightly sealed, and cells kept in PBS at 4°C until downstream library preparation. Cells were permeabilised by incubation in Lysis buffer 1 (10 mM Tris-HCL pH 8, 10 mM NaCl, 1 mM EDTA, 0.2% Triton X-100, pH 8 at 4˚C) for one hour at rt, followed by incubation in Lysis buffer 2 (10 mM Tris-HCL, 150 mM NaCl, 1 mM EDTA, 0.3% SDS, pH 8 at 25˚C) for one hour at 37˚C. Permeabilised cells were washed three times in 1x CutSmart Buffer (NEB) and blunt-end repaired using NEB Quick Blunting Kit +100 µg/mL BSA in a final volume of 50 µL at rt for one hour. Cells were then washed three times in 1x CutSmart Buffer and A-tailed using NEBNext dA-Tailing Module in a final volume of 50 µL at 37˚C for 30 mins. A-tailed cells were washed three times in 1x CutSmart buffer then incubated in 1x T4 DNA Ligase Buffer (NEB) for 5 mins at rt. A-tailed ends were labelled by ligation using T4 DNA ligase (NEB) + 0.4 µM Modified P5 adapter in a final volume of 50 µL at 16˚C for 16-20 h. Following ligation, excess P5 adapter was removed by washing cells 10 times in wash buffer at rt (10 mM Tris-HCL, 2 M NaCl, 2 mM EDTA, 0.5% Triton X-100, pH 8 at 25˚C), incubating for 2 mins each wash step. Cells were washed once in PBS and then once in nuclease free H2O. Genomic DNA was extracted by incubating cells in DNA extraction buffer (10 mM Tris-HCL, 100 mM NaCl, 50 mM EDTA, 1.0% SDS, pH 8 at 25˚C) + 1 mg/mL Proteinase K in a final volume of 100 µL for 5 mins at rt. The cell lysates were transferred to 1.5 mL Eppendorf RNA/DNA LoBind tubes and incubated at 65˚C for 1 hour, shaking at 800rpm. DNA was purified using Genomic DNA Clean & Concentrator-10 (Zymo Research), and eluted using 100 µL Elution Buffer. Genomic DNA was fragmented to 300-500bp using a Bioruptor Sonicator, and size selected using SPRI beads (GC Biotech) to remove fragments <150bp.  Fragmented and size-selected DNA was end-repaired using NEBNext Ultra II DNA Module. Fragmented and end-repaired DNA was added directly to the ligation reaction using NEBNext Ultra II Ligation Module according to the manufacturer's instructions using 7.5 µM Modified half-functional P7 adapter and omitting USER enzyme addition. The ligated sequencing libraries were purified using SPRI beads. Libraries were purified twice more using SPRI beads, and size selected to remove fragments <200bp to remove residual adapter DNA. Final clean libraries were quantified by qPCR using the KAPA Library Quantification Kit for Illumina Platforms. INDUCE-seq