GSM7426758: pre-cultured; Mus musculus; RNA-Seq
Freshly harvested spleens from chronically LCMV-infected micee were dissociated mechanically using gentle force with iron strainer and 3ml syringe. Transfer the homogenized cell solution into the 50ml conical tube and spin down at 1500rpm for 5min at RT. Aspirate the supernatant, resuspend the pellet in 2ml ACK buffer, and incubate it for 5min at RT to lyse red blood cells. After the incubation, add 10ml of R2 (2%FBS in RPMI media) and pour the splenocytes into 70um cell strainer to filter the solution. After spinning down the tube at 1500rpm for 5min, cells were stained with PE PD-1, APC GP33/GP276, APC-Cy7 CD8 antibodies, and Amcyan live/dead viability dye in FACS buffer (2% FBS and 2mM EDTA in PBS). Alive PD-1+GP33+GP276+ CD8 T cells were sorted using a FACS Aria II (BD Bioscience). Library was performed according to the manufacter's instructions (single cell 3' v3.1 protocol, 10x Genomics). Briefly, GCs were resuspended in the master mix and loaded together with partitioning oil and gel beads into the chip to generate the gel bead-in-emulsion (GEM). The poly-A RNA from the cell lysate contained in every single GEM was retrotranscripted to cDNA, which contains an Ilumina R1 primer sequence, Unique Molecular Identifier (UMI) and the 10x Barcode. The pooled barcoded cDNA was then cleaned up with Silane DynaBeads, amplified by PCR and the apropiated sized fragments were selected with SPRIselect reagent for subsequent library construction. During the library construction Ilumina R2 primer sequence, paired-end constructs with P5 and P7 sequences and a sample index were added.