title: GSM8838968: siControl Vehicle Replicate 2; Homo sapiens; OTHER
library_construction_protocol: Following the indicated transfections and optional 18 hr BRACO19 treatment, cells were harvested, resuspended in PBS, and plated at a density of 2 x 105 cells per well onto a poly-D-lysine 96-well plate. The plates were spun at 300 x g followed by incubation for 2 hr at 37°C and 5% CO2. Cells were then fixed by adding methanol-free formaldehyde at a 4% concentration and incubating for 10 min. After washing twice with PBS, the cells were stored in 200 μL PBS at 4°C until the INDUCE-seq was performed as previously described. Briefly, cells were permeabilized with Buffer 1 (1hr 4°C; 10 mM Tris-HCl, 10 mM NaCl, 1 mM EDTA, 0.2% Triton x-100) and Buffer 2 (1hr 37°C; 10 mM Tri-HCl, 150 mM NaCl, 1 mM EDTA, 0.3% SDS) with intervening PBS washes. Cells were washed with CutsmartTM buffer (NEB, B6004S) then DNA breaks were end-repaired (1 hr room temperature; Quick Blunting Kit (NEB, E1201L) plus 100 µg/mL BSA) and A- tailed (30 min 37°C; NEBNext® dA-Tailing Module; NEB, E6053L) with intervening CutsmartTM washes. A modified p5 adapter was ligated onto A-tailed break-ends in-situ using T4 DNA ligase (16-20 hr 16°C; NEB, M0202M). Excess adapter was removed with 10 washes with wash buffer (10 mM Tris-HCl, 2 M NaCl, 2 mM EDTA, 0.5% Triton x-100), then cells were lysed in DNA extraction buffer (10 min, 37°C; 10 mM Tris-HCl, 100 mM NaCl, 50 mM EDTA, 1% SDS with addition of 1 mg/ml Proteinase K) and crosslinking was reversed (1 hr, 65°C). Genomic DNA was extracted and purified (ZR-96 Genomic Clean & Concentrator-5, Zymo), then sheared to a 300-500 bp (Covaris ME220) and size selected using CleanNGS beads (GC Biotech, CNGS-0050). DNA was end-repaired and A-tailed (NEBNext Ultra II End Repair/dA-Tailing Module, NEB E7546L) and a modified p7 adapter was ligated (NEBNext Ultra II Ligation Module, NEB E7595L) to repaired break-ends. Libraries were purified twice, and size selected using CleanNGS beads (GC Biotech, CNGS-0050). Purified libraries were quantified using qPCR (KAPA Library Quantification Kit for Illumina® Platforms (Roche, 07960255001) and 1 x 75 bp sequencing performed using a high output flow-cell on a NextSeq 550 (Illumina).
identifiers.namespace: GEO
identifiers.id: GSM8838968_r1