title: GSM8491123: Bulk ATAC-seq, GBM22, tumor; Homo sapiens; ATAC-seq
library_construction_protocol: 10,000-50,000 cells were washed with cold ATAC-seq resuspension buffer (RSB; 10 mM Tris-HCl pH 7.4, 10 mM NaCl, and 3 mM MgCl2 in water), and permeabilized with ATAC-seq lysis buffer (RSB supplemented with 0.1% NP40, 0.1% Tween-20, and 0.01% digitonin). For the transposition step, nuclei were resuspended in 50 µl of transposition mix (25 µl 2× TD buffer, 2.5 µl transposase, 16.5 µl PBS, 0.5 µl 1% digitonin, 0.5 µl 10% Tween-20, and 5 µl water) and incubated at 37 °C for 30 min in a thermomixer with shaking at 1,000 r.p.m. The DNA fragments were cleaned up with the Qiagen MinElute PCR Purification kit (Qiagen, Cat. 28004) and PCR amplified for 5 cycles with Illumina Nextera adaptors using NEBNext High Fidelity 2x Master Mix (New England Biolabs, Cat. M0541S). Next, a qPCR reaction was performed using 10% of the pre-amplified PCR product. We looked for the Ct value at ¼ maximum fluorescence to determine the number of additional cycles to amplify the ATAC-seq libraries. After a second round of PCR amplification (all together, a total of 8-10 cycles), we performed a second cleanup with 1.8x Ampure XP beads (Aline biosciences, Cat. C-1003-50).