Ultra high-throughput single-cell transcriptome and epitope profiling of healthy human PBMCs using SCITO-seq2

This study presents comprehensive single-cell transcriptome and surface protein profiling using SCITO-seq2, an enhanced platform that improves the recovery of unique transcripts while enabling simultaneous detection of surface proteins. The analysis was conducted in two phases: first, using healthy donor PBMCs to validate technical performance, and second, by comparing samples from healthy controls, patients with childhood systemic lupus erythematosus (cSLE), and patients with CTLA4 haploinsufficiency and autoimmune infiltration (CHAI). These results demonstrate the platform's applicability to both normal and disease states. Overall design: The study consisted of two major experimental sets. PBMC 1: Fresh PBMCs from a healthy donor were divided into four replicate pools, each labeled with pool-specific antibodies and RNA probes. After pooling, 200,000 cells were loaded onto a Chromium Next GEM Chip Q for GEM generation to assess data consistency across pools derived from the same sample. PBMC 2: Fresh PBMCs were isolated from healthy donors (n=4), childhood systemic lupus erythematosus (cSLE) patients (n=4), and CTLA4 haploinsufficiency with autoimmune infiltration (CHAI) patients (n=2). For sample multiplexing, cells from healthy and cSLE groups were labeled with unique TotalSeq-B hashing antibodies and pooled within their respective groups, while CHAI samples were processed as individual pools. All samples underwent fixation and RNA probe hybridization following the 10x Genomics protocol. For each experimental set, 200,000 cells were loaded onto a Chromium Next GEM Chip Q for GEM generation. Libraries were prepared using the 10x Genomics Fixed RNA Profiling protocol and sequenced on an Illumina NovaSeq 6000 platform with 2 × 150 bp paired-end reads.