GSM8900571: Single-cell TCR V(D)J of Homo sapiens: ATL patient 3; Homo sapiens; RNA-Seq
Blood samples were collected from ATL patients, and peripheral blood mononuclear cells (PBMCs) were isolated using Ficoll-Paque Plus (Cytiva). The isolated PBMCs were subsequently cryopreserved in liquid nitrogen for future analysis. Single-cell gene expression and TCR immune profiling were performed using the Chromium Next GEM Single Cell 5' Reagent Kits v2 Dual Index (10x Genomics), following the manufacturer's protocols. Initially, thawed patient PBMCs were washed with complete RPMI medium supplemented with 10% FBS, counted, and resuspended in 1X PBS + 0.04% BSA at a final concentration of 1,000–1,200 cells/μl. GEMs were generated using Chromium Next GEM Single Cell 5ʹ Gel Bead Kit (Cat # PN-1000267) in the Chromium Controller using the Chromium Next GEM Chip K Single Cell Kit (Cat # PN-1000287). After GEM formation, the gel bead was dissolved and any co-partitioned cell was lysed. The transcripts present in the beads were barcoded and reverse-transcribed into full-length cDNA using Chromium Next GEM Single Cell 5' GEM Kit v2, (Cat # PN-1000266). The 10x barcoded, full-length cDNA was amplified by PCR, and the size distribution and the DNA concentration of the amplified cDNA were assessed using the Agilent Bioanalyzer High Sensitivity dsDNA kit. The amplified full-length cDNA was subsequently used for V(D)J amplification using the Chromium Single Cell Human TCR Amplification Kit (Cat # PN-1000252). Finally, both the 5' gene expression and the TCR V(D)J libraries were prepared using the Library Construction Kit (Cat # PN-1000196) following the manufacturer's protocols. The resulting libraries were sequenced using the DNBSEQ-G400RS platform.