GSM8023390: dual-CAR triple-Switch rep1 [single cell]; Mus musculus; RNA-Seq
Following stimulation, T cells were stained for CD4 and CD8 and hash-tagged according to CAR/switch-receptor configuration (TotalSeq-B anti-mouse hashtag antibodies B0301, B0302, B0303; Biolegend 155831, 155833, and 155835) and sorted for viable, DAPI-negative, CD4+ and CD8+ T cells using a BD FACSAriaII. T cells were sorted into PBS + 2% BSA and were then washed twice in PBS 0.04% BSA and resuspended at ~ 1000 cells/μL. Sorted hash-tagged CD4+ and CD8+ T cells were combined at 1:1 ratios for each CAR/switch-receptor configuration sample. Each combined CD4+/CD8+ hash-tagged CAR T cell configuration was pooled 1:1:1 (dual-CAR, dual-CAR dual-Switch, dual-CAR triple-Switch) and approximately 30,000 cells were submitted for single-cell sequencing per combined biological replicate sample. Following quality control (QC) steps, the single cell suspension was introduced into the Chromium Next GEM Chip G (10X Genomics PN 1000120) and gel bead in emulsion generation, cDNA synthesis, cDNA amplification, and library preparation of 30,000 cells were carried out using the Chromium Next GEM Single Cell 3' Kit v3.1 (10X Genomics PN 1000268) according to the manufacturer's instructions. cDNA amplification included 11 cycles and 138-159 ng of the material were used to prepare sequencing libraries with 10-12 cycles of PCR. Equimolar quantities of indexed libraries were pooled and sequenced on a NovaSeq X in a PE28/88 paired-end run using the NovaSeq X 10B Reagent Kit (100 cycles) (Illumina).  Average reads generated per cell were 23,000. cDNA and feature barcodes tagged with cell barcodes and unique molecular identifiers were generated using methods described above. Smaller feature barcode fragments were separated from longer amplified cDNA using a 0.6X cleanup using AMPure XP beads (Beckman Coulter catalog # A63882). A 3' Feature Barcode Kit (10X Genomics PN 1000276) was used to construct libraries according to the manufacturer's protocol with 10 cycles of PCR. Equimolar quantities of indexed libraries were pooled and sequenced on a NovaSeq X in a PE28/88 run using the NovaSeq X 10B Reagent Kit (100 cycles) (Illumina). An average of 335 million paired reads was generated per sample.