GSM6594953: bulk CD8+ T cells, dirty, timestamped at day 1, rep 2 (ATAC-seq); Mus musculus; ATAC-seq
FACS sorted cells were permeabilized in lysis buffer (10 mM Tris-Cl pH 8.0, 300 mM sucrose, 10 mM NaCl, 2 mM MgAc2, 6 mM CaCl2, 0.2% NP-40 (Igepal), 0.5 mM DTT, 1x Pierce protease inhibitor (Thermo Scientific), and 40 units of RiboLock RNase inhibitor (Thermo Scientific) per 10 mL buffer) by brief aggressive pipetting, washed with buffer W (10 mM Tris-Cl pH 8.0, 300 mM sucrose, 10 mM NaCl, 2 mM MgAc2, 0.008% Tween20, 0.5 mM DTT, 1x protease inhibitor, and 40 units of RiboLock RNase inhibitor per 10 mL buffer), and resuspended in storage buffer (50 mM Tris-Cl pH 8.3, 40% glycerol, 5 mM MgCl2, 0.1 mM EDTA, 0.5 mM DTT, 1x protease inhibitor, and 40 units of RiboLock RNase inhibitor per 10 mL buffer). The cells were pelleted at 1000 g for 8 minutes for each step of buffer change. The permeabilized cells were counted, snap-frozen in liquid nitrogen, and stored at -80°C. Libraries were prepared by the TREx core at Cornell University from 25,000 permeabilized cells as described in Buenrostro et al. (2015) and Corces et al. (2017). Briefly, frozen permeabilized cells were thawed, washed twice in ATAC-RSBbuffer containing 0.1% Tween20, resuspended in Transposition Mix (Nextera DNA Sample Preparation Kit, Illumina) containing 0.1% Tween20, resuspended in 25 µL Omni-ATAC Transposition Mix (10 mM Tris pH 8, 5 mM MgCl2, 10% DMF, 0.1% Tween20, 0.01% digitonin, 1.25 µL TDE1 enzyme (Illumina), and 8.25 µL PBS in final 25 µL volume per sample), incubated for 30 minutes at 37°C with shaking (900 rpm), with immediate addition of Zymo binding buffer. After Zymo column cleanup, libraries were amplified using Nextera PCR oligos (Nextera Index Kit, Illumina) and 2x HiFi Master Mix (New England Biolabs) with an initial incubation at 72°C for 5 minutes to extend the segmented ends and 11 PCR amplification cycles.  The libraries were cleaned up twice (in series) with a 2:1 ratio of SPRIselect beads (Beckman Coulter).