GSM8656051: EL4-NP68 specific memory CD8 T cell; Mus musculus; RNA-Seq
B6 mice were transferred with naive CD8 T cells from F5 transgenic mice. The next day, mice were hallenge with vaccinia virus (VV) or EL4 tumor cells, both expressing NP68 peptide. CD45.1+ F5 memory cells were sorted from the spleens of VV- or EL4-immunised mice 9 weeks after the primary challenge. Naive F5 cells were sorted from the spleen of F5 x CD45.1 mice. Total CD8 T cells were first enriched from splenocytes using an autoMACS® Pro Separator with the CD8a+ T cell isolation kit (Miltenyi Bio-tec) according to manufacturer's instructions. Enriched CD8 T cells were stimulated (VV-CD8_R, EL4-CD8_R, naive_R)  or not (VV-CD8, EL4-CD8, Naive) with NP68 peptide (10 nM) for 2h at 37 °C. F5 cells were then single-cell sorted according to their viability and their expression of CD45.1, CD8a, and CD44. Single cell dDNA were prepared using Smart-Seq2 protocol. Cells were sorted into a 96-well plate containing 4 uL of lysis solution (PBS, 0,04% Triton 0,4%, 2 U/uL RNaseOut [TFS], 2,5 uM of poly(T) reverse transcription primers and 2,5 mM dNTPs), on a FACS Aria II (BD Biosciences). Library construction was performed following the Smart-seq2 protocol. Briefly, mRNA was reverse transcribed into cDNA and pre-amplified by PCR. cDNAs was then fragmented by tagmentation and Illumina sequencing adaptors containing in-dexes allowing for cell identification were added. The cDNA was amplified by PCR. Each 96-cells libraries were pooled together and both quality and quantity were tested using  a DNA high sensitivity D1000 ScreenTape on Tapestation 4200 (Ag-ilent). Libraries were sequenced on an Illumina HiSeq in paired-ends (2 x 150 bp) at an average sequencing depth of ∼1M reads/cells.