Ia6 2

CD19+CD3−IgD−CD14−CD20−CD27+CD38++ plasmablasts were single-cell sorted (BD FACSAria) as described [19 ,24 ] (Supplementary Methods). PBMC-staining used fluorophore-conjugated antibodies against CD19 (HIB19; Biolegend), CD3 (UCHT1; BD), IgD (IA6-2; BD), CD14 (MφP9; BD), CD20 (L27; BD), CD27 (CLB-27/1; LifeTechnologies), CD38 (HB7; BD), IgA (IS11-8E10; Miltenyi), and IgM (MHM-88; Biolegend). PBMCs were co-stained with pooled citrullinated-peptide tetramers comprised of 14 citrullinated peptides (Supplementary Table 1) to identify ACPA-producing plasmablasts (Supplementary Methods).

Plasmablasts (CD19+CD3-IgD-CD14-CD20-CD27+CD38++) were single-cell sorted (BD FACSAria) from PBMCs as previously described [19 (link), 23 (link)], while all CD19+ B cells were single-cell sorted from ST. PBMCs and ST-derived cells were stained with fluorophore-conjugated antibodies against CD19 (HIB19; Biolegend or SJ25C1; BD), CD3 (UCHT1; BD), IgD (IA6–2; BD), CD14 (MφP9; BD), CD20 (L27; BD), CD27 (CLB-27/1; LifeTechnologies or M-T271; BD), CD38 (HB7; BD), IgA (IS11–8E10; Miltenyi), and IgM (MHM-88; Biolegend). PBMCs from samples isolated at Stanford were co-stained with pooled citrullinated-peptide multimers comprised of 14 citrullinated peptides to identify ACPA-producing plasmablasts [24 (link)].
Sequencing of immunoglobulin gene-encoded mRNA from individual B cells was performed using cell barcodes as previously described [19 (link), 23 (link)–25 (link)]. Unique well-specific oligonucleotide barcodes (TruGrade Oligos; IDT) were added during reverse transcription (Maxima Reverse Transcriptase; ThermoFisher Scientific). The cDNA from all 96 wells were pooled and HC and LC genes were amplified using gene-specific PCR primers, followed by paired-end sequencing (2×330) using Illumina MiSeq. Samples were prepared using two [25 (link)] or three [24 (link)]rounds of PCR amplification with gene-specific primers.