title: GSM5008756: mild symptomatic COVID-19 donor, 201657790 (GEX); Homo sapiens; RNA-Seq
library_construction_protocol: Cryopreserved PBMCs were thawed before being slowly transferred to a 50 ml conical tube and diluted in PBS containing 0.5 mM EDTA buffer (PEB). The cells were then centrifuged at 300 x g for 5 minutes at 4°C, washed twice and resuspended in 0.5 ml PEB. B cells were isolated from PBMCs by Human Pan B cell immunomagnetic negative selection kit (StemCell Technologies, MA) per manufacturer's instructions. Cell concentration was determined using trypan blue staining with a TC20 automated cell counter (Biorad, CA) and adjusted if necessary, to achieve the desired concentration of 700-1200 cells/µl. About 7,000 purified B-cells from each sample were combined with Chromium Next GEM Single Cell 5ʹ Gel Beads v1.1 and Reverse Transcription master mix (10x Genomics, CA) to generate barcoded Gel beads in emulsion (GEM) according to the manufacturer's recommendations. The GEMs were recovered and purified using silane magnetic beads (Thermo Fisher Scientific, MA). The full-length cDNA along with cell-barcode identifiers were PCR amplified for 11 cycles and then purified using SPRISelect magnetic beads (Beckman Coulter, CA). The purified cDNA was inactivated in 80% ethanol before being removed from BSL-4. The quality of ethanol precipitated DNA was assessed by Bioanalyzer and both B-cell receptor (BCR) and 5' gene expression (GEX) libraries were prepared as per manufacturer's instructions. Libraries were quantified, both GEX and BCR libraries were pooled according to equivalent molar concentrations and sequenced on Illumina NovaSeq6000 sequencing platform. BCR and 5'GEX