title: GSM2976730: virtual memory CD8 cells timestamped at day 28, rep 1 [ATACseq]; Mus musculus; ATAC-seq
library_construction_protocol: CD8+ cells were magnetically enriched with CD8 microbeads and sorted to > 95% purity with a FACS Aria III on CD4- CD8+ TdTomato+ CD44 hi (virtual memory) or lo (true naïve) populations. Cells were permeabilized in lysis buffer (10mM Tris-Cl, 300mM sucrose, 10mM NaCl, 2mM MgAc2, 3mM CaCl2, and 0.1% NP-40(Igepal)) for 10mins, washed, resuspended in storage buffer (50mM Tris-Cl, 40% glycerol, 5mM MgCl2, and 0.1mM EDTA), counted, snap-frozen, and stored at -80°C until further use. Libraries were prepared from 30,000 permeabilized cells primarily as described in Buenrostro et al, 2015 (Curr Protoc Mol Biol) and Corces et al, 2017 (Nature). Briefly, frozen permeabilized cells were thawed, washed twice in ATAC-RSBbuffer containing 0.1% Tween20, resuspended in Transposition Mix containing 0.1% Tween20 and PBS, and incubated for 30min at 37deg with shaking (1000rpm). After column cleanup, libraries were amplified using Nextera PCR oligos (Illumina) and Ultra II Q5 Master Mix (NEB) for a total of 12-14 PCR cycles based on qPCR quantification after the intial 5 cycles. Post-amplification, libraries were cleaned up twice (in series) with a 2:1 ratio of SPRIselect beads (Beckman Coulter).
attributes.tag: GEO Accession
attributes.value: GSM2976730