GSM6943745: PBMC, S1, TCR V(D)J-enriched cDNA; Homo sapiens; RNA-Seq
Cryopreserved PBMC from SARS-CoV-2-naive (n = 4) or convalescent (n = 4) donors collected approximately 10 days post vaccination were thawed in sterile-filtered RPMI 1640 (Thermo Fisher Scientific) with 10% FBS (Hyclone) and 0.2% Benzonase (MilliporeSigma) and then centrifuged at 300xg for 10 minutes at room temperature. Viable cells were counted using the Guava easyCyte. Cells were centrifuged at 300xg for 10 minutes at room temperature and plated at a density of 2x106 167 cells/well in a 96-well U-bottom plate. Next, cells were blocked with 1 ug/mL antiCD40 (clone HB14, Miltenyi Biotec) at 37degreeC. The following day, cells were stained with Fixable Aqua viability dye (Invitrogen) for 30 minutes at room temperature. An antibody cocktail was prepared for sorting with the following markers: antiCD7 FITC (clone CD7-6B7, BioLegend), antiCD69 BV711 (clone FN50, BioLegend), antiCD137 APC/Fire 750 (clone 4B4-1, BioLegend), and antiCD40L/CD154 PE-Cy5 (clone TRAP1, BD Biosciences). Additionally, eight cocktails were prepared with the following TotalSeq-C antibody-derived tags (ADTs): antiCD3 (clone UCHT1, BioLegend), antiCD4 (clone RPA-T4, BioLegend), antiCD8a (clone RPA-T8, BioLegend), antiGD (clone B1, BioLegend), antiTRAV1-2 (clone 3C10, BioLegend), antiCD62L (clone DREG-56, BioLegend), antiCCR7 (clone G043H7, BioLegend), antiCD45RA (clone HI100, BioLegend), antiCD28 (clone CD28.2, BioLegend), antiCD127 (clone A019D5, BioLegend), antiCD95 (clone DX2, BioLegend), antiCXCR3 (clone G025H7, BioLegend), antiCXCR5 (clone J252D4, BioLegend), antiCCR4 (clone L291H4, BioLegend), antiCCR5 (clone J418F1, BioLegend), antiCCR6 (clone G034E3, BioLegend), antiCD25 (clone BC96, BioLegend), antiCD38 (clone HIT2, BioLegend), antiCD26 (clone BA5b, BioLegend), antiHLA-DR (clone L243, BioLegend), antiCD161 (clone HP-3G10, BioLegend), antiCD103 (clone Ber-ACT8, BioLegend), antiCD14 (clone M5E2, BioLegend), antiCD16 (clone 3G8, BioLegend), antiCD56 (clone 5.1H11, BioLegend), antiCD11b (clone ICRF44, BioLegend), antiCD11c (clone S-HCL-3, BioLegend), antiCD169 (clone 7-239, BioLegend), antiPD185 L1 (clone 29E.2A3, BioLegend), antiCD19 (clone HIB19, BioLegend), antiCD20 (clone 2H7, BioLegend), antiCD163 (clone GHI/61, BioLegend), antiCD86 (clone IT2.2, BioLegend), antimouse IgG2a isotype control (clone MOPC-173, BioLegend), antimouse IgG1 isotype control (clone MOPC-21, BioLegend), antimouse IgG2b isotype control (clone MPC-11, BioLegend). One of the following TotalSeq-C anti-human hashtag oligonucleotides (HTOs) were also included in each of the TotalSeq cocktails for cell hashing: Hashtag 1 (clones LNH-94/2M2, BioLegend), Hashtag 2 (clones LNH-94/2M2, BioLegend), Hashtag 3 (clones LNH-94/2M2, BioLegend), Hashtag 4 (clones LNH-94/2M2, BioLegend), Hashtag 5 (clones LNH-94/2M2, BioLegend), Hashtag 6 (clones LNH-94/2M2, BioLegend), Hashtag 7 (clones LNH-94/2M2, BioLegend), and Hashtag 8 (clones LNH-94/2M2, BioLegend). Cells were stained with the sorting antibody cocktail and the appropriate TotalSeq cocktail for 30 minutes at 4degreeC. Next, cells stimulated with S1 or S2 peptides were separately pooled together and passed through a 35 um cell strainer. Activated T cells defined as Live/Dead-, CD7+, CD69+ and CD137+ or CD154+ were sorted using a BD FACSAria III Cell Sorter. Two wells of a Chromium Next GEM Chip G were loaded with either the pooled S1-activated or S2-activated T cells. The Chromium Next GEM Single Cell V(D)J Reagent kit (v1.1, 10x Genomics) was used to prepare mRNA, TCR, and surface protein libraries. cDNA amplification and target enrichment were quantified using a Qubit 3 Fluorometer (Invitrogen) and assessed for quality using a 4200 TapeStation System (Agilent). Libraries were constructed and sequenced to a depth of approximately 11,900 reads per cell using the NextSeq 500 system (Illumina).