GSM7329949: Batch2-2_Target; Homo sapiens; OTHER
PBMCs were isolated from whole blood with BD vacutainer CPT tubes (BD), resuspended in CELLBANKER 1 (Takara), and stored in -80°C. PBMC thawed as described above were resuspended with Cell Staining Buffer (BioLegend) supplemented with Human TruStain FcX. After 2-3 minutes, 2x AbSeq master mix of Immune Discovery Panel (BD Biosciences) prepared as the provider's protocol was added to the cell suspension, and stained for 15 minutes on ice. Then, the antibody mix 3 (Supplementary Table S1) with Sample Tag oligonucleotide-conjugated antibodies (Single-Cell Multiplexing Kit, BD Biosciences) was added to the cell suspension and left on ice for 15 minutes for staining. Stained cells were washed with Cell Staining Buffer. Non-naive CD4+ and CD8+ cells (ID2 and ID27) or only non-naive CD8+ cells (ID10, ID19, ID22, ID31, and ID35) were sorted using FACS Aria II or Aria III and FACSDiva software (v8.0.1). Nonviable cells were excluded from the analysis based on forward and side scatter profiles and PI staining. Data were analyzed using FlowJo software (version 10.8.1). Live cells were stained with Calcein AM (Nacalai) and counted using Flow-Count fluorospheres and a Cytoflex flow cytometer. About 25,000 - 30,000 labeled cells were loaded on a BD Rhapsody cartridge (BD) and processed using BD Rhapsody™ Cartridge Reagent Kit (BD) following the manufacturer's instructions. Single-cell mRNA, AbSeq barcodes, and Sample Tag barcodes were captured by BD Rhapsody beads coated with poly(T) oligonucleotide with a unique cell barcode and molecular barcode on each bead. Single-cell cDNA synthesis was performed using BD Rhapsody cDNA Kit (BD) following the manufacturer's protocol. Targeted mRNA, TCRseq, and Abseq Index libraries were prepared using Immune Response Panel HS (BD) according to the manufacturer's instructions with the following modifications. (1) We used the original set of primers for TCR amplification, as summarized in Table S2. (2) We performed additional PCRs to introduce a unique dual index for sequencing (3rd PCR for mRNA and TCR), as described in Table S3. (3) We used ProNex Size-Selection DNA purification System (Promega) for the purification of PCR products, as summarized in Table S8. Amplified libraries were quantified using a KAPA SYBR Fast qPCR Kit (KAPA Biosystems), and size distribution was analyzed by Microchip Electrophoresis System MultiNA (Shimadzu). Targeted RNAseq, TCRseq, and AbSeq libraries were sequenced on an Illumina Novaseq 6000 S4 flowcell (67 bp read 1 and 140 bp read 2) using NovaSeq 6000 S4 Reagent Kit v1.5 to a depth of approximately 20,000 reads, 7,000 reads, and 10,000 reads per cell, respectively.