title: GSM3693106: CCRF-CEM (ATAC-seq); Homo sapiens; ATAC-seq
library_construction_protocol: ATAC-seq experiments for the Jurkat, MOLT-4, CCRF-CEM, RPMI-8402 and K-562 cell lines were performed using the Omni-ATAC-seq method. In each experiment, 1 x10^5 cells were centrifuged at 1000 x g for 10 min at 4 ºC. Following aspiration, a cell count of the supernatant was performed, the remaining cell number was calculated, and all further reagents in the protocol were titrated to this cell number. Library construction was conducted using the Omni-ATAC-seq method.  For every 5 x 10^5 cells, nuclei were isolated in 50 ul cold ATAC-Resuspension Buffer (RSB) (10 mM Tris-HCl pH 7.4, 10 mM NaCl, 3 mM MgCl2) containing 0.1% NP40, 0.1% Tween-20, and 0.01% Digitonin, and pipet-mixed up-and-down at least 5 times. Nuclei isolation mix was incubated on ice for 3 exactly minutes, washed in 1 ml of cold ATAC-RSB containing 0.1% Tween-20 (but no NP40 or Digitonin) and centrifuged at 1000 x g for 10 min at 4 ºC. Nuclear DNA was tagmented in 50 ul Transposition mix (25ul 2 × TD buffer, 2.5ul transposase (100nM final), 0.5 ul 1% digitonin, 0.5 ul 10% Tween-20, 16.5 ul PBS and 5 ul diH2O), and incubated in a thermomixer at 37°C, 1000 x g for 30 min. Tagmented DNA was purified with Zymo DNA Clean and Concentrator-5 Kit (cat# D4014). Library amplification was performed using custom indexing Nextera primers from IDT in a 50 ul Kapa Hi Fi Hot Start PCR reaction (cat# KK2602). Following 3 initial cycles 1 ul of PCR reaction was diluted in 1 ml buffer (10mM Tris-HCl pH 8.0, 0.05% Tween) and 4 ul of each dilution were run in triplicate 20uL quantitative PCR (Kapa qPCR Library Quantitation Kit cat# KK4824) to calculate the optimum number of final amplification cycles. Library amplification was followed by SPRI size selection with Kapa Pure Beads (cat# KK8002) to retain only fragments between 80-1,200 bp. Library size was obtained on an Aglient Bio-Analyzer or TapeStation using a High Sensitivity DNA kit and factored into final Kapa qPCR results to calculate the final size-adjusted molarity of each library. ATAC-seq libraries were pooled by size adjusted molarity and sequenced to obtain a minimum of 30 to 50 million reads from each cell line in paired-end 42 x 42 confirugration.
attributes.tag: GEO Accession
attributes.value: GSM3693106