GSM7957352
To extract thymocytes for both single-cell RNA sequencing (scRNAseq) and flow cytometry, the thymus tissue was placed in complete RPMI 1640 media (Gibco, #22400-071) (10% heat-inactivated fetal bovine serum (FBS, Sigma-Aldrich), 1% non-essential amino acids (Sigma-Aldrich), 1% Sodium Pyruvate (Sigma-Aldrich), 1X GlutaMAX (Gibco), 1% Penicillin/Streptomycin (Gibco), and 1X 2-mercaptoethanol (BME, Sigma-Aldrich)), cut into small pieces, and gently pressed with the back of a 10 ml syringe to release thymocytes. The resulting suspension was passed through a 70 µm filter. Thymocytes and PBMCs were isolated using a Ficoll-Paque density gradient provided by Cytiva. PBMCs were cryopreserved in FBS with 10% DMSO from Sigma-Aldrich and stored in liquid nitrogen. Tetramer staining for MAIT and iNKT cells was performed on freshly isolated thymocytes. For tetramer staining of MAIT and iNKT cells, freshly isolated thymocytes were used. To enrich for thymic MAIT and thymic/blood iNKT cells, up to 2x109 cells were incubated with MR1-5-OP-RU-PE-Tet or CD1d-PBS57-PE respectively in MACS buffer (0.5% BSA, 2mM ETDA, PBS), for 25 mins at room temperature. Cells were washed twice and incubated with anti-PE microbeads (Miltenyi), followed by separation using an autoMACS Pro Separator (Miltenyi) according to manufacturer's instructions. Enriched MAIT and iNKT from thymus, enriched iNKT from PBMC, unenriched gd T ,CD4+ and CD8+ from thymus, and unenriched MAIT, gd T , CD4+ and CD8+ T cells from PBMC were stained with the following cell surface markers in MACS buffer at room temperature for 20 mins: CD3-AF488 (clone OKT3, Biolegend), CD14-eFluor450 (clone 61D3, ThermoFisher), CD19-eFluor450 (clone H1B19, ThermoFisher), Va7.2-BV785 (clone 3C10, Biolegend), Va24-PerCP-Cy5.5 (clone C15, Biolegend), CD4-AF710 (clone OKT4, Tonbo), CD8a-PE-Cy7 (clone SK1, Tonbo), TCR-BV650 (clone 11F2, BD Biosciences), FcgR block (Miltenyi). Cells were washed twice and resuspended in MACS buffer prior to cell sorting on the Aria 3 (BD Biosciences). Single cell whole transcriptomes and TCR sequencing libraries were prepared using the BD Rhapsody Single-Cell Analysis System (BD Biosciences) according to the manufacturer's specifications. Prior to cell sorting on the Aria 3 (BD Biosciences) and during cell surface antibody staining, up to 2 x106 enriched or unenriched cells were labeled with an oligonucleotide-tagged antibody sample tag (BD Biosciences). From infant thymus and PBMC donors up to 5 populations were sorted after doublet, viability, B cell (CD19+CD3-) and monocyte (CD14+CD3-) discrimination: 1. MAIT cells (MR1-5-OP-RU-Tet+Va7.2+CD3+), 2. iNKT cells (CD1d-PBS57-Tet+Va24+CD3+), 3. gd T cells (CD3+TCRgd+), 4. CD4+ T cells (CD4+CD8-CD3+) and CD8+ T cells (CD8+CD4-CD3+). Prior to cDNA library preparation for the WTA and VDJ libraries, all cell subsets from the different donors were pooled, with up to 12 unique sample tags combined per library. Libraries were quantified and pooled according to equivalent molar concentrations and sequenced on the NovaSeq sequencing platform at the University of Colorado Genomics Core with the following read lengths: read 1 – 150 cycles; read 2 – 150 cycles; and i7 index - 8 cycles.