title: GSM4725415: PCOS-day12-rep3; Homo sapiens; ATAC-seq
library_construction_protocol: At days 0, 3, and 12, cells were trypsinized and 100,000 cells were subject to nuclei preparation for ATAC-Seq. ATAC-seq libraries were generated according to Berostro et al. (doi: 10.1002/0471142727.mb2129s109) with minor modifications. Nuclei were treated with Tn5 transposon TDE1 (Nextera DNA Library Prep kit, Illumina, San Diego, CA) for 35 min at 37°C. The transposition reaction was then purified using Zymo DCC-5 columns (Zymo Research, Irvine, CA). Following, Polymerase Chain Reaction (PCR) was performed using 25 μl NEBNext High-Fidelity 2x PCR Master Mix (NEB, MA, Boston) 4 µl of each Index 1 (i7) and index 2 (i5) (Nextera XT Index kit, Illumina, San Diego, CA), 2 µl of PPC (Illumina, San Diego, CA) and 15 µl of purified tagmentation reaction. After performing the initial extension and 5 PCR cycles, quantitative PCR was performed on 5 µl of the previous PCR reaction by adding 1 µl of SYBR Green (15x), 1 µl of PPC, 5 μl NEBNext High-Fidelity 2x PCR Master Mix and 3 µl of H2O in a final volume of 15 µl for 20 cycles. The number of additional cycles was determined as the cycle number corresponding to ¼ of maximum fluorescent intensity. Final libraries were purified using 45 µl (1:1 ratio) of SPRI beads (Beckman and Coulter, Indianapolis, Indiana) cat# B23318). Final libraries were visualized on a TapeStation 2200 using a D1000 ScreenTape. Libraries were pooled and paired-end sequenced (PE 2X100) on the Illumina HiSeq4000.