GSM8263350: femaleKOTCR; Mus musculus; RNA-Seq
Freshly collected splenocytes were sorted for CD3+CD4+ T cells. Sorted CD4+ T cells were stimulated for 6 hours in RPMI + 10% FBS (v/v) + 1x Pen/Strep with anti-CD3/anti-CD28 Dynabeads for 6 hours in 37°C incubator. Library construction was performed according to the manufacturers instructions (Single Cell 3' v3.1 Protocol, 10x Genomics). Briefly, CD4+ T cells were resuspended in the master mix and loaded with partitioning oil and gel beads into the chip to generate the gel beads-in-emulsion (GEM). The poly-A-RNA from the cell lysate was then retrotranscribed to cDNA. The pooled barcoded cDNA was cleaned with Silane Dynabeads and amplified by PCR. SPRIselect reagent was used to select appropriately sized fragments for library construction. During library construction, Ilumina TruSeq Read 2 primer sequence, paired-end constructions with P5 and P7 sequences and a sample index were added.