3482151116 NGS-immunoglobulins.xlsx

Peripheral blood mononuclear cells (PBMCs) were resuspended in staining buffer (PBS supplemented with 1% fetal bovine serum) and stained with mouse-anti-human CD27-AF647 (AbD Serotec), mouse-anti-human CD19-PB (AbD Serotec) and goat-anti-human IgM-AF488 (Life Technologies) for 1 hour at 4°C, protected from light. Cells were washed, resuspended, and stained with propidium iodide (Sigma-Aldrich) at a final concentration of 10 ug/mL. Cells gated CD19+ were sorted on BD FACSAria Fusion flow cytometer (BD Biosciences).Sorted B cells were resuspended in RNA extraction buffer (QuickExtract RNA Extraction Kit, Lucigen). cDNA was generated using the Maxima H Minus cDNA Synthesis Master Mix (Thermo Fisher) using 5 uL of extraction buffer per 10 uL reaction as per manufacturer’s protocol. 2 uL of cDNA product was directly used in 20 uL reactions with Platinum Taq Master Mix as per manufacturer’s protocol for separate amplification of the heavy and light chain variable regions. Illumina adaptor and barcode sequences added by further PCR with Q5 Hot Start Hi-Fidelity Master Mix and the PCR products purified with AMPure XP magnetic beads. Purified PCR products were pooled in the same molar ratio as the number of B cells from each of the four samples, diluted to 8 pM and sequenced on the Illumina MiSeq with 2x300 bp kit with 25% PhiX spike in. Individual reads were separated by sample and analyzed with MIXCR to derive unique clonotypes based on CDR1 and 3 homology. The corresponding germline sequences were retrieved from IMGT, the international ImMunoGeneTics database®.