GSM9285554: Cd8+ P14 cells (young), young mouse; Mus musculus; RNA-Seq
Whole liver was dissected out and placed on a 40 or 70 um cell strainer mounted on 50 mL tube. Single cell suspension was obtained by mashing using 3 mL syringe plunger while passing 10 % FBS in RPMI. Liver samples were centrifuged ar 60 r.c.f., 4 degC for 2 min with no brake to pellet hepatocytes. The supernatant was collected and centrifuged at 420 r.c.f, 4 degC for 4 min. Immune cells were enriched with 40 % Percoll (Cytiva) in HBSS. The pellet was resuspended in ACK lysis buffer (KD Medical) for red blood cell lysis. The the lysis buffer was neutralised and removed by centrifugation. Cells were then counted with trypan blue and haemocytometer. Immune cells from 7 young and 7 old mice were combined to enrich for adoptively transferred P14 CD8+ T cells. First, T cells were enriched using Invitrogen T cell enrichment kit (Invitrogen 8804-6822). The enriched T cells were then stained for the congenic marker Thy1.1, CD8, DAPI. Antigen-specific response was confirmed by anti-PD1 staining observed during sorting. Cells were sorted out using flow cytometer at the SBP flow core. All Thy1.1+ CD8+ T cells were collected in 0.05% BSA in PBS. Cells were counted for viability and then loaded onto 10X scRNAseq Chip. For single-cell sequencing, T-cell suspensions were processed using the GEM-X Universal 3' Gene Expression v4 kit (10x Genomics, Pleasanton, CA, USA; Cat. Nos. 1000686, 1000690, 1000215) according to the manufacturer's user guide (CG000731, Rev B). Single-cell libraries were sequenced on the Element Biosciences AVITI platform (San Diego, CA, USA) using the Cloudbreak FS High Output kit (Cat. No. 860-00015) with paired-end reads of 28 bp for Read 1 and 90 bp for Read 2. Raw sequencing data were processed with Cell Ranger (vX.X, 10x Genomics) using the mm10 (GRCm38) mouse reference genome to generate gene–cell count matrices for downstream analysis.