Single nucleus mRNA library generated from pooled samples of rheumatoid arthritis PBMCs as part of a 10x single cell multimodal ATAC and Gene Expression experiment with FACS population hashtags.
From rheumatoid arthritis PBMC samples from 4 patients, we enriched for CD4 T cells using the MACS protocol and sorted for 4 populations using FACS (CD4+CD127-CD25hi Tregs, CD4+CD127-CD25int Tregs, CD4+CD25-PD1+CXCR5- TPH, CD4+CD25-PD1+CXCR5+ TFH). Nuclei were isolated using an adaptation of the manufacturer's protocol (10X Genomics). Each sorted population was tagged with a hashing antibody before pooling across populations. Approximately thirty thousand transposed nuclei were loaded on Chromium Next GEM Chip J followed by GEM generation. 10x Barcoded DNA from the transposed DNA (for ATAC), 10x Barcoded, full-length cDNA from poly-adenylated mRNA (for Gene Expression), and 10x Barcoded, cDNA from Hashtag oligos (for Protein Antibody Hashtags) were produced during GEM incubation. The ATAC libraries and Gene Expression/Hashtag libraries were then generated separately. Both ATAC and Gene Expression library types were sequenced to an average of 30,000 reads per cell on different flowcells with recommended sequencing cycles according to the manufacturer's protocol (Chromium Next GEM Single Cell Multiome ATAC + Gene Expression, 10X Genomics). HTO libraries were sequenced at approximately 5K reads per cell.