title: GSM8036101: OA13-A; Homo sapiens; ATAC-seq
library_construction_protocol: Freshly sorted cells carrying breast cancer samples were treated with trypsin to obtain 50,000 cells (counted by using trypan blue exclusion) for ATAC-seq. After a wash step in cold cell wash buffer (CWB; 10 mM Tris-HCl pH 7.4, 10 mM NaCl, 3 mM MgCl2), outer membranes were disrupted in lysis buffer (CWB + 0.1% NP40) on ice for 2 minutes. The lysis reaction was stopped with the addition of 1 ml CWB. After a centrifugation step at 1,500g for 10 minutes, pelleted nuclei were used for the next step. In a 50-μl final volume, tagmentation was performed for 30 minutes at 37℃ using the Nextera DNA library prep kit (Illumina FC-121-1030). After adding SDS to a final concentration of 0.2%, DNA was purified on AMPure XP beads (Beckman Coulter A63881) using a 2:1 ratio (v/v) of beads:tagmented DNA. Freshly eluted DNA was barcoded and amplified in 110-μl PCR volume (NEB Next Q5 Hot Start HiFi PCR, M0543L) to generate a library with the following PCR program: 65℃, 5 minutes; 98℃, 30 seconds; 11 cycles of (98℃, 10 seconds ramping to 65℃, 30 seconds), 4℃ hold. Quality control of the libraries was performed with a Bioanalyzer 2200 (Agilent Technologies, D1000 screentapes and reagents, 5067-5582) to assess size range of amplified DNA fragments and with Quant-iT PicoGreen dsDNA Assay Kit (Thermo Fisher P11496) to quantify the DNA fragments generated. ATAC libraries were then pooled at equimolar concentration and were sequenced multiplexed on the Illumina HiSeq with 50-bp paired-ends.