GSM8965013: sample2_tet+ WT_2, d32_2, d123_2_GEX; Mus musculus; RNA-Seq
Prior to fluorescence-activated cell sorting (FACS), cells from each individual mouse were stained with hashtag oligonucleotides (HTOs) to enable multiplexing and improve sample throughput. T cells sorted from spleens of non-coding mRNA or OVA mRNA vaccinated mice. Single cells were encapsulated in Gel-Beads-in emulsion (GEMs) using the 10X controller with a GEM-X Single Cell V(D)J Reagent Kit (5' chemistry; 10x Genomics) following the manufacturer's protocol. Libraries were quantified using the Qubit dsDNA High Sensitivity (HS) Assay Kit (Invitrogen) and quality-checked using a High Sensitivity DNA chip on a Bioanalyzer 2100 system (Agilent Technologies). The sequencing was carried out on an Illumina NovaSeq 6000 system using an S4 flow cell with paired-end reads (2 × 150 cycles). Raw reads from scRNA-seq libraries were aligned to the mouse reference genome mm10-2020-A using Cell Ranger v9.0.0 (10x Genomics). Filtered feature-barcode matrices, containing only high-confidence cellular barcodes, were used for downstream analysis. Single-cell TCR reads were aligned to the mouse reference genome vdj_GRCm38_alts_ensembl-7.0.0. Only productive TCRα and TCRβ chains were retained for subsequent analyses.