Fluidigm preamp master mix

cDNA was synthesized with the SuperScript IV VILO Master Mix with ezDNase Enzyme kit (Thermo Fisher Scientific; cat. no. 11766050). Enrichment for the targets of interest was performed with the Fluidigm Preamp Master Mix (Fluidigm; cat. no. 100-5581) and the same set of primers to be used for real-time PCR. TaqMan quantitative PCR assays were then performed on a BioMark HD system (Fluidigm; cat. no. BMKHD-BMKHD), with the TaqMan Fast Advanced Master Mix (Thermo Fisher Scientific; cat. no. 4444556) and the FLEXsix IFC Gene Expression Kit (Fluidigm; cat. no. 100-6309) for BLNK, BTK, CCL3, CCL4, CD19, CD79A, CD79B, CR2, IL10, LYN, PRDM1 (BLIMP-1), SYK, and TLR7.

Total RNA was purified from sorted cell populations using the RNeasy Micro Kit (Qiagen), and concentration was determined using the Quant-IT RiboGreen RNA assay kit (Thermo Fisher Scientific). First-strand cDNA synthesis was performed with a High Capacity cDNA Reverse Transcription Kit (Thermo Fisher Scientific) followed by preamplification of genes of interest using the Fluidigm PreAmp Master Mix (Fluidigm Europe B.V.) with 25 ng of total RNA and in accordance with the manufacturer’s instructions. Exon-spanning primers to amplify genes of interest were designed using Primer-Blast (see Table 2 for details). To increase sensitivity, genes of interest were preamplified by 12 cycles of PCR using pooled assays followed by exonuclease I treatment (New England Biolabs) to remove unincorporated primers. Final preamplified cDNA was diluted 1:5 in Tris-EDTA buffer. Gene expression analysis was performed using the Biomark HD system from Fluidigm (Fluidigm Europe B.V.) in accordance with the manufacturer’s instructions and standard settings. Data were analyzed using the Real-Time PCR Analysis Software (Fluidigm Europe B.V.), and the resulting cycle threshold values were normalized to Ppia to obtain delta CT values.