GSM5839858: AG14_SIV_CD4_28plus_Mem; Chlorocebus pygerythrus; ATAC-seq
Cells were isolated from fresh peripheral blood by density gradient centrifugation using lymphocyte separation media (MP Biomedicals, #0850494-CF), washed in PBS, counted, and approximately 5e6 cells resuspended in 100ul PBS. Cells were stained with LIVE/DEAD amine-reactive viability dye (Thermo Fisher Scientific, #L34966) and a panel of antibodies including anti-CD3 alexa fluor 700 (clone SP43-2, BD Biosciences, #557917), anti-CD4 allophycocyanin (clone L200, BD, #551980), anti-CD8α pacific blue (clone RPA-T8, BD, #558207), CD28 energy-coupled dye [ECD] (clone 28.2, Beckman Coulter, Brea, CA, #6607111), CD95 Cy5-PE (clone DX2, BD, #561977), for 20 minutes at 4°C. Cells were washed in PBS and resuspended in complete RPMI with 10% FBS. For each population, 30,000 cells were sorted using a FACSymphony S6 cell sorter (BD). Tagmentation was performed by pelleting 30,000 sorted cells and resuspending in 2x Tagmentation Buffer (Illumina, #20034198), 0.02% Digitonin, and 0.1% Tween-20, with Tagment DNA TDE1 Enzyme (Illumina) and incubating at 37°C for one hour. The mix was then incubated with proteinase K for 30 minutes at 40°C. High molecular weight DNA was removed with a 0.7x selection ratio of Agencourt AMPureXP beads (Beckman Coulter, #A63881) and low molecular weight DNA was isolated from the remainder with a 1.2x selection ratio. Library amplification was performed with KAPA HiFi HotStart ReadyMix (KAPA, #KK2601) and i5 and i7 indexing primers (Nextera DNA CD Indexes, #20018708) for 11-15 total cycles depending on the sample concentration reached after 5 cycles (KAPA Library Quantification kit, #KK4835). Library DNA was purified with a 1x selection ratio of AMPureXP beads. The final library was verified by High Sensitivity DNA Bioanalyzer (Agilent, #5067-4626) to ensure the presence of appropriately sized DNA fragments.