title: GSM8673672: T47D ATAC, DMSO, biol rep2; Homo sapiens; ATAC-seq
library_construction_protocol: ATAC-seq libraries were generated following a modified Omni-ATAC-seq protocol (PMID: 28846090). Following treatment, both control and treated samples were washed, trypsinized and counted in a hemocytometer. To check viability, cells were counted with 0.4% trypan blue. Cell viability >85% is required. After counting, 100,000 cells were pelleted at 500 RCF for 5 min at 4°C and lysed on ice for 3 min with ATAC-Resuspension Buffer (10mM Tris-HCl pH 7.4, 10mM NaCl and 3mM MgCl2) containing 0.1% Tween-20 (Fisher cat no. BP337), 0.1% IGEPAL® CA-630 (Sigma cat no. I3021), 0.01% Digitonin (ThermoFisher Scientific cat no. BN2006). After incubation with lysis buffer, cell lysates were washed with ATAC-Resuspension Buffer containing 0.1% Tween-20. After washing, nuclei were pelleted at 500 RCF for 10 min at 4°C. Pelleted nuclei was resuspended in transposition mix containing the Illumina Tagment DNA TDE1 Enzyme (Tn5) and Buffer (Illumina cat no. 20034197), 0.1% Tween-20 and 0.01% Digitonin, and incubated in a thermomixer at 1000 RPM for 30 min at 37°C. After tagmentation, fragments were cleaned using MinElute PCR Purification Kit (Qiagen cat no. 28004). Cleaned up fragments were resuspended in ultrapure water. The transposed fragments were then amplified with NEBNext High-Fidelity 2X PCR Master Mix (New England BioLabs cat no. M0541S) and indexed with Illumina Nextera XT Index Kit (Illumina cat no. FC-131-1001). Final libraries were cleaned up and size selected with AMPure XP SPRI Reagent (Beckman Coulter cat no. A63880). To assess library quality, 1ul of each sample was submitted to Agilent Bioanalyzer at Queen's University Department of Pathology and Molecular Medicine. Finally, samples were sent to Genome Quebec Innovation Center and sequenced on an Illumina NovaSeq6000 platform using a 100 base-pair paired-end read set-up.