GSM8155073: WT, Pan, 100, 24hr, D12, 3dpi, Rep2, ATAC; Homo sapiens; ATAC-seq
Samples were collected on differentiation day 9 and day 12 with or without 8-hour or 24-hour NEUROG3 induction. Cells were dissociated with Accutase into single cell suspension. 50,000 cells were collected for lysis and transposition based on Omni-ATAC protocol. Briefly, cells were lysed with Lysis buffer (10mM Tris-HCL, 10mM NaCl, 3 mM MgCl2, 0.1% NP-40, 0.1% Tween-20, 0.01% Digitonin), washed with washing buffer (10mM Tris-HCL, 10mM NaCl, 3 mM MgCl2, 0.1% Tween-20), and incubated with Transposition buffer (Nextra) for at 1000 rpm 37°C for 30 minutes. DNA was purified (Qiagen MinElute) for library construction Library was prepared and sequenced at Kottyan lab, CCHMC. 5ul of the amplified DNA was then used to perform qPCR to determine the optimal number of additional cycles to prevent amplification saturation of DNA libraries. Double size selection of amplified libraries (0.5x – left sided, 1.8x – right sided) was performed using AMPure XP beads and the DNdA was eluted in a final volume of 20ul in 0.1x TE buffer. Purified libraries were sequenced on an Illumina NextSeq 500 instrument with a 2 3 75bp read length.