GSM7315487: IgD+ B, 16C301, week -1, VDJ-derived cDNA; Macaca mulatta; RNA-Seq
Frozen rhesus macaque PBMCs 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 DNase I (MilliporeSigma), followed by one wash with R10 and one wash with FACS buffer (PBS with 2% FBS). For B and innate cell staining, cells were resuspended in 100µL of Live/Dead Fixable Blue Dead Cell Stain Kit (Invitrogen, cat# L23105) diluted 1:200 in PBS for 10 min at room temperature. Cells were washed with FACS buffer and incubated for 20 min with the staining cocktail consisting of antibodies and probes. Additionally, each sample was labelled with 1µl of TotalSeq-C Hashtag antibodies (Biolegend) that was incubated together with the staining cocktail. After incubation, cells were washed twice with FACS buffer and resuspended in R10 for sort.  From each sample, 10,000-60,000 innate cells were sorted into a tube containing FBS according to the gating strategy showed in Fig. S1B. From baseline samples, 50 thousand naïve B cells were also sorted in a separate tube. From week 2 and 6 samples, antigen-specific cells were single-cell sorted into 96-well plates containing 5 µL of TCL buffer (Qiagen) with 1% b-mercaptoethanol for sequencing by SmartSeq. All sorts were performed using a BD FACSymphony S6 Cell Sorter (BD Biosciences) with BD FACSDiva Software version 9.5.1 (BD Biosciences) and data were analyzed using Flowjo v10.8.1. Frozen rhesus macaque PBMC 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 DNase I (MilliporeSigma) and rested for 1 hour at 37oC / 5% CO2.  Cells were stimulated with two peptide pools corresponding to S1 and S2 of the vaccine insert SARS-CoV-2 S protein (JPT Peptide Technologies) at 2 µg/mL of each peptide for 6 hours at 37oC / 5% CO2.  A DMSO only control was included for each sample.  Anti-CD154 BV421(Biolegend, clone 24-31, cat# 310824) was included during the 6-hour culture and GolgiStop (BD Biosciences) was added after 2 hours of stimulation.  Following stimulation, cells were washed and stained with Aqua LIVE/DEAD dye (ThermoFisher) for 10 minutes, and subsequently stained with antibody mix. In addition, cells were incubated with TotalSeqTM Hashtag antibodies (Biolegend) with separate antibodies applied to the DMSO only condition and the stimulated conditions. For each animal, 1000-2000 memory CD4 cells from the DMSO only condition were sorted and combined with memory cells expressing CD154 and CD69 from each S pool stimulation for processing for 10X Genomics. Bulk-sorted cells were pooled into a tube and loaded on the 10x Genomics Chromium Chip according to the manufacturer's protocol for the Next GEM Single Cell 5' Kit v1.1 (10x Genomics, PN-1000165). To sequence single-cell gene expression and cell surface oligonucleotides (from Hashtag antibodies), libraries were prepared according to manufacturer's instructions using the Chromium Single Cell 5' Library Construction Kit (10x Genomics, PN-1000020) and Chromium Single Cell 5' Feature Barcode Library Kit (10x Genomics, PN-1000080), respectively. To sequence TCR repertoire libraries were prepared using the Chromium Single Cell V(D)J Enrichment Kit (10x Genomics, PN- PN-1000005). These libraries were sequenced on an Illumina NovaSeq platform (Illumina). To sequence BCR repertoire, heavy and light chains were amplified from the cDNA using IgG_REV, IgA_REV, IgK_REV or IgL_REV primers (Table S3) with the addition of Illumina sequences as detailed in (25). These Illumina-ready libraries were sequenced using 2x300 paired-end reads on an Illumina MiSeq Full-length transcriptomes were sequenced using a modified SMART-Seq protocol. Heavy and light chain variable regions were enriched by amplifying cDNA with a forward primer complementary to the TSO and IgA/IgG_REV or IgK/IgL_REV primer pools. OTHER: scRNA-seq with FeatureSeq and scAIRR-seq