GSM9099925: Jurkat_8B10_VAV1_CRISPRa_HIVTargeting_rep_1; Homo sapiens; ATAC-seq
Approximately 200,000 cells from each Jurkat T cell clone underwent Ficoll density  gradient centrifugation for dead cell removal (Ficoll-Paque PLUS Medium, VWR #95038-168). Approximately 50,000 cells per aliquot (3 replicates per Jurkat T cell clone) were used for ATAC-seq and 50,000 cells per aliquot for RNA-seq. For ATAC-seq, aliquots 50,000 cells were resuspended in 50 µL ATAC-lysis buffer (10 mM Tris-HCl at pH 7.4, 10 mM NaCl, 3 mM MgCl2, 0.1% Tween-20, 0.1% NP-40, 0.01% digitonin) on ice for 3 min. We then diluted the mixture with 1mL ATAC-wash (10 mM Tris-HCl at pH 7.4, 10 mM NaCl, 3 mM MgCl2, 0.1% Tween-20). Cells were washed and pelleted then transposed for 30 min at 37˚C (1× TD buffer with 33% PBS, 0.1% Tween-20, 0.01% digitonin, 2.5 µL TDE1). After transposition, DNA was isolated using Qiagen MinElute PCR cleanup kit, #28004. Library prep was performed using NEBNext Ultra II Q5 master mix (NEB #M0544X) and Nextera indexed according to NEBNext Ultra II (NEB #E7645) instructions. Following baseline amplification, typically 8 cycles, SYBR qPCR was used to determine total cycle numbers needed. Typically, a total of 12 additional PCR cycles of library amplification were added, depending on qPCR results. After validating library size on an Agilent TapeStation, 150-bp paired-end sequencing was performed by NovaSeq 6000 at Yale Center for Genome Sequencing.