GSM8001338: 54_C2D1; Homo sapiens; OTHER
PBMCs were thawed by immersing the cryotube in a 37°C water bath until a small ice shard remained. The cryotube contents were then mixed using a 1 mL pipette and transferred to a 50 mL centrifuge tube. Additionally, 1 mL of media was mixed with the cryovial and then transferred to the same 50 mL tube. Four sequential dilutions were performed by incrementally adding media to the centrifuge tube while gently agitating the mixture. Specifically, 2 mL, 4 mL, 8 mL, and 16 mL were added in succession, with a 30-second interval between each step. The PBMCs were subsequently centrifuged at 400g for 5 minutes. The supernatant was removed, leaving approximately 1 mL, in which the cells were resuspended. An additional 9 mL of media was added, followed by another 5-minute centrifugation at 400g. The supernatant was once again removed and discarded. The cells were finally resuspended in 1xDPBS+0.04% BSA. Cell count and viability were assessed using a Cellometer K2 and Acridine Orange/Propidium Iodide (AO/PI) staining. Single cell libraries were constructed using the 10x Genomics chromium 5′ v2 workflow according to the manufacturer's protocol. All experiments and conditions aimed for a capture of approximately 6000 cells. Libraries were quantified with Qubit and checked for fragment size using the TapeStation D1000 kit (Agilent). Libraries were pooled in equimolar concentration for a total pooled concentration of 1.8 nM. The libraries were sequenced using S4 (200 cycle) sequencing kits on the Illumina NovaSeq 6000 with a read one length of 100 cycles, and a read two length of 100 cycles. TCR libraries were constructed from the cDNA generated for each sample during the 5' v2 workflow according to the manufacturers protocol (CG000331 REV A). The TCR libraries were then quantified using the QUBIT DNA HS kit (cat# Q32854) and fragment size determined using the TapeStation D1000 kit (Agilent). The libraries were then pooled in equimolar concentration for a total pooled concentration of 2nM. The libraries were sequenced using SP (100 cycle) sequencing kits on the Illumina NovaSeq 6000 with a read one length of 26 cycles, and a read two length of 90 cycles.