title: GSM8653145: SARS-CoV-2-specific B cells, donor2/3/6, mRNA-derived cDNA; Homo sapiens; OTHER
library_construction_protocol: Cryopreserved PBMC samples were thawed into warm R10 media (RPMI + 10% Fetal Bovine Serum + 2 mL L-Glutamine, 100 U/ml penicillin and 100 µg/ml streptomycin; all reagents from Gibco) containing Benzonase nuclease (MilliporeSigma), followed by one wash with R10 and one wash with stain buffer (BD Pharmingen Stain Buffer containing 0.2% bovine serum albumin, BD Biosciences). After washing twice with R10 and once with stain buffer, cells were resuspended in 50µL of Live/Dead Fixable Blue Dead Cell Stain Kit (Invitrogen) diluted 1:200 in DPBS (Gibco) for 5 min at room temperature, then 5µL of Human TruStain FcX (Biolegend) and 1µl of Totalseq-C hashtag antibody (Biolegend) were added to each sample and incubated for additional 10 min on ice. Cells were washed with stain buffer and incubated on ice for 30 min with 100µL of the stain cocktail consisting of fluorochrome- and oligonucleotide-labeled antibodies and fluorochrome-labeled probes (SARS-CoV-2 proteins WA1 S-2P BV711, WA1 RBD BV570, WA1 NTD BUV737, Omicron BA.1 S-2P APC, Omicron BA.1 RBD BV421, and Omicron BA.1 NTD BUV737). Antigen-specific IgG+ or IgA+ B cells were sorted into a tube containing 1 mL of R10 using a BD FACSymphony S6 cell sorter. After sort, cells were pelleted at 1500 rpm for 10 min at 4º C, resuspended in 30µL and loaded into the 10x Genomics Chromium Chip according to the manufacturer's protocol for the Chromium Next GEM Single Cell 5' Reagent Kit v2 Dual Index (10x Genomics). To sequence single-cell gene expression and cell surface oligonucleotides libraries were prepared according to manufacturer's instructions using the Chromium Single Cell 5' Library Construction Kit (10x Genomics, PN-1000190) and Chromium Single Cell 5' Feature Barcode Library Kit (10x Genomics, PN-1000541). These Illumina-ready libraries were sequenced using 2x100 cycles paired-end reads on an Illumina NovaSeq. To sequence IG repertoire, heavy and light chains were amplified from the cDNA generated for gene expression using IgG_REV, IgA_REV, IgK_REV or IgL_REV primers (Lima et al, Nat Commun, 2022; Schramm et al, Nat Commun, 2023) with the addition of Illumina sequences. These Illumina-ready libraries were sequenced using 2x300 cycles paired-end reads on an Illumina MiSeq.