GSM5506043: A673 ETV6 dTAG cells - 72 hour dTAGV-1 treated - ATAC-seq - replicate 1; Homo sapiens; ATAC-seq
ATAC-seq was performed using a publicly available protocol (https://www.med.upenn.edu/kaestnerlab/assets/user-content/documents/ATAC-seq%20Protocol%20(Omni)%20-%20Kaestner%20Lab.pdf). 100,000 cells from each sample were washed in PBS and resuspended in 1.5 ml reaction tubes in 50 µl Lysis Buffer (48.5 µl Resuspension Buffer, 0.5 µl 10% NP-40, 0.5 µl 10% Tween-20, 0.5 µl 1% Digitonin; Resuspension Buffer was comprised of 10 mM 1M Tris-HCl pH 7.5, 10 mM NaCl, 3 mM MgCl2). The lysis reaction was incubated for 3 minutes on ice. To each sample, 1 ml of Wash Buffer (990 µl Resuspension Buffer, 10 µl 10% Tween-20) was added and tubes were inverted a few times. Cells were pelleted by centrifugation at 500xg for 10 minutes at 4°C. The supernatant was discarded such that intact nuclei in the pellet were isolated. The nuclei were resuspended in 50 µl Transposition Reaction Mix, comprised of 25 µl 2X Tagment DNA Buffer and 2.5 µl Tagment DNA Enzyme 1 (Illumina Inc., # 20034197), 16.5 µl PBS, 0.5 µl 10% Tween-20, 0.5 µl 1% Digitonin, and 5 µl H2O. Samples were incubated at 37°C for 30 minutes on a thermomixer at 1,000rpm. Following incubation, DNA from each sample was extracted using a reaction cleanup kit (Qiagen, # 28204) and DNA was eluted using 10 µl Elution Buffer. DNA was PCR-amplified for 5 cycles. 5 µl from each partially amplified library was used to perform qPCR to determine the required number of additional PCR cycles, calculated as the number of cycles needed to reach 1/3 of the maximum R in each qPCR reaction. Samples were amplified an additional 3 or 4 PCR cycles. Libraries were purified using a double-sided Agencourt AMPure XP bead purification (Beckman Coulter, # A63880), first at 0.5X the reaction volume, conserving the supernatant, and then at 1.3X the reaction volume. Purified libraries were eluted in 20 µl H2O. The molarity of each library was estimated by measuring the DNA concentration using the Qubit dsDNA Broad Range Assay kit (Thermo Fisher Scientific, # Q32850) and by analyzing the distribution of DNA fragment sizes using the Tapestation D5000 dsDNA assay (Agilent; ScreenTape, # 5067-5588; Ladder, # 5067-5590; Reagents, # 5067-5589). Each library was diluted to 4 nanomolar and libraries were pooled in equal volumes. Libraries were sequenced in 37-basepair paired-end fashion for 75 cycles, aiming for >60 million reads per sample, on the Illumina NextSeq 500 instrument.