GSM9335743: RiSi05_Hu_1_CD4_9D_Gex; Homo sapiens; RNA-Seq
According to the previously described AIM assay protocol, PBMCs were thawed and stimulated for 24 h with the S MP. Cells were washed and stained with an antibody cocktail containing the antibodies described above for 30 min at 4°C, protected from light. TotalSeqTM-C oligonucleotide-conjugated antibodies (BioLegend, San Diego, CA) were also added at this step at 0.01mg/mL final concentration (one distinct antibody per sample). After two washes in PBS, cells were resuspended into 500 μL of FACS buffer (PBS containing 2mM EDTA (pH 8.0) and 0.5% BSA) and stored at 4°C until flow cytometry acquisition. S-specific CD4+ and CD8+ AIM+ T cells were subsequently sorted using a BD FACSAria Fusion cell sorter (Becton Dickinson), as previously described26. On average 20,000–40,000 AIM+ T cells per subject were sorted for single-cell RNA-seq assays (10x Genomics, Pleasantown). Cells were collected directly into low-retention, sterile 1.5 mL collection tubes (Thermo-fisher) pre-chilled on ice and containing 500 μL of PBS:FBS solution (1:1, vol:vol) supplemented with RNAse inhibitor (1:100, Takara Bio). 5 to 6 different samples were multiplexed, each labeled with distinct TotalSeq-C DNA-oligo barcoded, totaling approximately 60,000 cells per 10X lane (equivalent to one well of the 10X chip). Samples were centrifuged at 600 ×g for 10 min at 4°C, and the supernatant was carefully removed, leaving behind 10–12 μL of residual volume. Cell pellets were resuspended in 25 μL of 10x Genomics-compatible resuspension buffer (0.22 μm filtered PBS containing 0.04% ultrapure bovine serum albumin, Sigma-Aldrich). 33 μL of the resuspended cells was transferred to an 8-strip PCR tube for downstream processing as per the 10x Genomics protocol. Remaining cells were used for a cell-counting quality control. Following the manufacturer's recommendations, single-cell RNA libraries were generated using the 10x Genomics standard 5′TAG v2 chemistry. Both cDNA amplification and library preparation were carried out using 13 PCR cycles. Barcoded cDNA products were collected, quantified, and pooled at equimolar concentrations. The libraries were sequenced on the Illumina Novaseq6000 platform with paired-end sequencing (S4 100x100cycle, Illumina) configured as follows: read length: read 1, 100 cycles; read 2, 100 cycles; i7 index, 10 cycles and i5 index 10 cycles. Size and quantity quality controls were performed throughout the procedure by capillary DNA high sensitivity electrophoresis (HS NGS Fragment Kit, 1-6000bp, Fragment Analyzer, Agilent) and Picogreen assay (Quant-iT PicoGreen dsDNA Assay Kits and dsDNA Reagents). Sequencing depth was aimed to reach 30,000 reads per cell for gene expressions, 8,000 reads/cell for TCR, and antibody-feature sequencing (multiplexing analysis).