title: GSM7213572: TWIST1FV, long-term dTAG, rep3; Homo sapiens; ATAC-seq
library_construction_protocol: DNase-treated cells were harvested by Accutase, counted using the Countess II (Invitrogen), and 50,000 cells were collected by centrifugation at 500g for 5 min at 4°C. Cells were resuspended in lysis buffer (resuspension buffer (RSB), or 10 mM Tris-HCl pH 7.4, 10 mM NaCl, and 3 mM MgCl2, with 0.1% Igepal CA-630, 0.1% Tween-20, and 0.01% digitonin) for 3 min, then quenched by dilution with RSB with 0.1% Tween-20. Lysate was centrifuged for 10 min at 500g at 4°C and then resuspended in transposition buffer (25 ul TD buffer and 2.5 ul TD enzyme (Illumina, 20034197), 16.5 ul PBS, 0.01% digitonin, 0.1% Tween-20, and water up to 50 ul). Transposition reactions were performed for 30 min at 37°C, and then cleaned up with the DNA Clean & Concentrator-5 kit (Zymo, D4013) and eluted in 21 ul 10 mM Tris-HCl pH 8. Transposed DNA was pre-amplified 5 cycles with NEBNext Ultra II Q5 master mix (New England Biolabs, M0544) with a cycling protocol of 72°C for 5 min, 98°C for 30s, and 5 cycles of 98°C for 10s, 63°C for 30s, 72°C for 1 min. Then 5 ul of the 50 ul reaction was used to run a qPCR reaction (with the same cycling protocol except the initial 72°C incubation) to determine the optimal number of PCR cycles for each sample. The remaining portion of the reaction was then amplified the appropriate number of cycles, and then subjected to two rounds of double-sided Ampure XP bead cleanup, with 0.5x/1.3x and 0.5x/1.0x bead ratios (numbers indicate bead ratios added in first and second steps). Libraries were quantified by Qubit dsDNA high sensitivity assay (Invitrogen, Q33231), run on a 5% polyacrylamide TBE gel to check the size distribution, and then pooled for sequencing.