GSM7093526: ATACseq -KO15 - day 5 + XAV; Mus musculus; ATAC-seq
Although viability of the cells is high (> 90-95%), we treated them in suspension nonetheless with 0.1 mg/ml DNase I (Worthington, LS002005) for 30 minutes at 37 ºC directly in the medium and added digestion buffer (0.5 mM CaCl2, 2.5 mM MgCl2). Cells were washed with PBS and incubated for 5 minutes with TrypLE which was subsequently quenched with medium. We used the omni-ATAC protocol to prepare ATACseq libraries as described before (Buenrostro et al., 2013; Corces et al., 2017).  After two washes with ice-cold PBS, 50,000 cells were pelleted in a pre-cooled 1.5 mltube by centrifugation for 5 minutes at 600 rcf at 4 ºC in a microcentrifuge. Supernatant was removed and 50 µl of ATAC-lysis buffer was added (10 mM Tris-HCl pH 7.4, 10 mM NaCl, 3 mM MgCl2, 0.1% NP40, 0.1% Tween-20, 0.01% digitonin) and incubated on ice for 3 minutes, followed by addition of 1 ml of ATAC-wash buffer (ATAC-lysis buffer, without NP40 and digitonin) and the tube was inverted 3 times. The nuclei were pelleted for 10 minutes at 600 rcf at 4 ºC in a microcentrifuge. Supernatant was removed, and nuclei were resuspended in 50 µl transposition reaction buffer (5 µl H2O, 15 µl PBS, 0.5 µl 10% Tween-20, 0.5 µl 1% digitonin, 25 µl TD buffer, 2.5 µl Tagment DNA Enzyme 1) (Tagment DNA Enzyme and Buffer Small Kit, Illumina, 20034197) and incubated for 30 minutes at 37 ºC at 1000 rpm in a thermomixer. The transposed DNA was cleaned up immediately following transposition ZymoDNAClean& Concentrator-5 kit (Zymo D4014) following manufacturer instructions. The transposed DNA was amplified in one pre-amplification step (5 cyles) and a final amplification (5 to 7 cycles) in the same 50 µl reaction using 1x NEBNext High-fidelty PCR master mix (Bioké/NEB, M0541S) and 125 nM of Ad1_noMX primer and Ad2.index primer with the following program: 72 ºC 5 min, 98 ºC 30 sec; 5 to 7x 98 ºC 10 sec, 63 ºC 30 sec, 72 ºC 1 min; 72 ºC 1 min. For each library, the optimal amplification was optimized using qPCR; 1/5th of the pre-amplified libraries was added to amplification master mix containing SYBR Green and run on a Applied Biosystems StepOnePlus Real-Time PCR system  for an additional 20 cycles. Relative log-fluorescence vs amplification cycle was plotted out to manually determine the appropriate amplification cycles. The number of cycles was selected to reach 1/3 of the final relative fluorescence unit value. Following amplification, the libraries were purified using ZymoDNAClean& Concentrator-5 kit (Zymo D4014) followed by 0.55x-1.75x dual size slection using AMPure XP beads. The quality of the libraries was assessed using an Agilent Bioanalyzer 2100 with the High Sensitivity DNA analysis kit (Agilent, 5067-4626) and concentration was determined using Qubit™ dsDNA HS Assay kit (Invitrogen, Q32854). The libraries were diluted to 4 nM and pooled together for SE50 sequencing on an Illumina Hiseq4000, aiming for minimally 25 million mapped and unique reads.