GSM8864238: TeloHAEC 72hr-Ctrl-SiRNA rep2; Homo sapiens; ATAC-seq
Approximately 250,000 cells were harvested from each biological replicate of TeloHAECs treated with ERG or Control SiRNA for 24 or 72 hours and cryopreserved in BAMbanker freezing media using a Mr. Frosty. On the day of ATAC-sequencing, cells were washed with 1 Ml of 37°C endothelial cell growth media (link) and pelleted at 500xg. Free DNA from cells lysed during the cryopreservation process was removed through a 30 minute incubation in endothelial cell growth media supplemented with 200 units of DNAse at 37°C. Approximately 50,000 live cells were isolated from the recovered population, and lysed in non-ionic detergent lysis buffer (10-mM Tris-HCl, pH 7.4, 3-mM MgCl2, 10-mM NaCl, 0.1% NP-40). Nuclei were pelleted and resuspended in 50 μl transposition reaction solution containing 2.5 μl Tn5 transposase attached to sequencing adapters (Illumina Nextera Tagment DNA enzyme TDE1, Nextera® DNA Sample Preparation Kit cat# FC-121-1030), 25 μl Tagment DNA buffer (20-mM Tris-HCl Ph7.6, 10-Mm MgCl2, 20% Dimethyl Formamide), 16.5 ul PBS, 5 ul NFH2O, 0.1% tween, and 0.01% Digitonin. Samples resuspended in Tn5 mixture were incubated at 37 °C for 30 min under gentle mixing conditions (300 rpm) using a thermomixer (Eppendorf, Hamburg, Germany). The transposed and adapter-ligated DNA fragments were purified using the Zymo Research DNA Clean and Concentrator purification kit and PCR amplified using barcoded primers (Nextera Index Kit, FC-121-1011), using cycle numbers determined by qPCR. A SageSciences PippinPrep was used to size select DNA between 125 and 600 basepairs using a 2% agarose gel. The final ATAC-seq libraries were sequenced with a 2 × 126-bp paired-end run in Illumina HiSeq2500 to obtain ~50 million, paired-end reads per sample.