title: GSM6640474: Sorted cells, CSP-derived cDNA [Pam1-cite-CSP11]; Homo sapiens; OTHER
library_construction_protocol: PBMCs were isolated the day after collection by density gradient centrifugation. If red blood cell contamination was present, cells were lysed with ACK buffer. Tonsil and adenoid tissues were stored in RPMI media with 5% FBS (VWR), gentamicin 50mg/mL (Gibco), and 1X antibiotic/antimycotic solution (Gibco) on ice immediately after collection. They were processed the next day, mechanically disrupted and filtered through a 100μm cell strainer to create a single cell suspension, lysed with ACK buffer and washed with PBS three times. The processed cells were stored in liquid nitrogen in the presence of FBS (VWR) with 10% DMSO. Banked PBMC, tonsils and adenoids from 2 donors with history of COVID-19 (CNMC 71 and 89) and one uninfected control (CNMC 99) were thawed from liquid nitrogen in a 37°C water bath for 2-3 mins. 2 mL of media consisting of RPMI with 10% of fetal bovine serum, 0.1mg/ml DNase I (Roche) and 10mM HEPES was added drop-by-drop to the thawed cells. Cells were further diluted by incremental addition of a 1:1 volume of media up to 8 mL, then centrifuged at 300 x g for 5 min. Cells were then resuspended in 300 μL of media, incubated at room temperature for 5 min, washed with media without DNase I, and filtered through a 100μm strainer before spinning down and resuspending in staining buffer (PBS + 1% BSA). Cells were then incubated with Fc blocker (Human TruStain FcX, BioLegend), stained with TotalSeq-C human hashtag antibodies (BioLegend) to uniquely label the sample origin (by tissue and donor), and washed with PBS + 0.04% BSA. Adenoids and tonsils from the 3 donors (6 samples in total) were pooled together and PBMCs from 3 were pooled together separately. The number of cells to pool from each tissue and donor was calculated with the aim of pooling a similar number of Spike1+ positive B cells from each sample. Pooled cells were first incubated with Fc blocker at 4°C for 10 min followed by CITE-seq and sorting antibody cocktails in the following order at 4°C: TotalSeq anti-CXCR3 antibody for 10 min, TotalSeq chemokine cocktail (anti-CCR7, CCR6, CXCR5 antibodies) for 10 min, and the rest of CITE-seq antibodies and fluorescence-labeled sorting antibodies and viability dye (Aqua) for 30 min. Cells were then washed with PBS+0.04% BSA and resuspended in PBS+2% FBS. S1+ and S1- B cells and CD95+ CD4+ and CD95+ CD8+ T cells were sorted from each pool on a BD FACS Aria Fusion sorter for tonsil/adenoid pool and FACS Aria Ill sorter for the PBMC pool. Cells were sorted into PBS+2% FBS. Library was prepared using Chromium Single Cell 5ʹ v2 (CG000330, Dual Index) kit (10x Genomics). V(D)J, mRNA and cell surface protein (CSP) were captured and indexed with a shared cellular barcode. Indexed cDNAs were then pooled and amplified by PCR according to the Chromium Single Cell 5' v2 protocol. SPRI bead size selection was performed in order to separate cDNAs from CSP Feature Barcode tagged DNA fragments. GEX libraries, CSP libraries and V(D)J libraries were separately constructed according to the manufacturer's instructions. Three types of libraries were pooled separately at the same molar concentration. The final pool composed of  80% GEX library, 10%V(D)J library and 10% CSP library for sequencing on NovaSeq 6000. Reference for feature surface markers was listed in Feature reference.xlsx.