title: GSM2337207: CD8_Acute-d27_Rep2; Mus musculus; OTHER
library_construction_protocol: All donor P14 cells were prepared from naive spleen using CD8 negative selection (Miltenyi Biotec). To generate effector, memory and exhausted CD8+ T cells, approximately 1-2 x 103 P14 cells were transferred into recipient mice, followed by LCMV infection one day later. Target effector/memory/exhausted P14 cells were harvested only from recipient spleens at d8 or d27 p.i. After enrichment using CD45.2 FITC Ab and anti-FITC microbes (Miltenyi), cells were stained and sorted by Aria II (BD). Naïve and effector/memory/exhausted CD8+ T (P14) cells were sorted by gating on CD8+ TCRVa2+ CD62L+ CD44lo and CD8+ TCRVa2+ CD45.2+ CD45.1- population, respectively. The following fluorochrome-conjugated antibodies from Biolegend were used for flow-cytometry: anti–TCR Vα2 (B20.1), anti-CD8α (53-6.7), anti-CD44 (IM7), anti-CD45.1 (A20), anti-CD45.2 (104), and anti-CD62L (MEL14). We sorted 40-50,000 cells per biological replicate (Naïve, Acute d8, Acute d27, Chronic d8, Chronic d27 and EL4), which were then washed once in cold PBS and lysed in 50μL cold lysis buffer (10 mM Tris-HCl, pH 7.4, 10 mM NaCl, 3 mM MgCl2, 0.1% IGEPAL CA-630). Lysed nuclei were incubated in Tn5 transposition reaction mix as described in (Buenrostro et al 2013) and purified using MinElute Reaction Cleanup kit (Qiagen). ATAC-seq fragments from one set of replicates for Acute d8, Acute d27, Chronic d8 and Chronic d27, as well as both biological replicates for Naïve cells and EL4 cells, were size selected for fragments between 115 and 600 bp using Pippin Prep 2% Agarose Gel Cassettes and the Pippin Prep DNA Size Selection System (Sage Science). Post size-selection, ATAC libraries were amplified and Nextera sequencing primers ligated using Polymerase Chain Reaction (PCR). Finally, PCR primers were removed using Agencourt AMPure XP bead cleanup (Beckman Coulter/Agencourt) and library quality was verified using a Tapestation machine. High quality ‘multiplexed’ DNA libraries were sequenced on the Illumina HiSeq2000. ATAC-Seq
attributes.tag: GEO Accession
attributes.value: GSM2337207