Rhapsody immune response targeted panel for human

A BD Rhapsody Single-Cell Analysis System (BD Biosciences) was used for the scRNA-seq analysis. In brief, PBMCs were processed into a single-cell suspension and loaded into a BD Rhapsody cartridge with >200,000 microwells. Then, a bead library was loaded into the microwell cartridge to saturation, and each cell was paired with a microbead. Next, the bead-cell complexes were hybridized with mRNA molecules to capture the barcoded oligos on the beads after lysing the cells in the microwell cartridge. The beads were collected into a single tube to generate a multiplex PCR-based library customized by the BD Rhapsody Immune Response Targeted Panel for Human (BD Biosciences). Fastq files were processed by an Illumina HiSeq 3000 platform and then processed into the expression matrix Fastq by the BD Rhapsody Analysis Pipeline. BD DataView software (BD Biosciences) and the R package Seurat (V 4.01) were used to analyze the expression matrix. The Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis was performed using the R package “enrichplot” (Yu et al., 2012 (link)). The raw expression data from these experiments are available at the NCBI Gene Expression Omnibus database, with the following identifier: GSE175429 (https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE175429).

CD45⁺ CD4⁺ T cells or Lin⁻ CD45⁺ CD127⁺ cells were isolated from blood samples using a FACSAria III cell sorter (BD Biosciences). Targeted scRNA-seq analysis was performed using the BD Rhapsody Single-Cell Analysis System (BD Biosciences), according to the manufacturer’s instructions. For the library construction, we used the BD Human Single-Cell Multiplexing Kit (#633781, BD Biosciences) and the BD Rhapsody Immune Response Targeted Panel for Human (#633750, BD Biosciences), which consisted of primer sets for 399 genes. Sequencing was performed using an Illumina HiSeq X (Illumina, San Diego, CA), and the fastq files were converted using BD Rhapsody Analysis Pipeline (BD Biosciences) and analyzed using the BD DataView software v.1.2.2 (BD Biosciences).