GSM7667184: Mdomestica_thymus_MAIT; Monodelphis domestica; RNA-Seq
A freshly isolated thymus was maintained in CO2-independent medium (Gibco) at 4°c until processing. Sample processing started less than 6 hours after euthanasia.  The thymus was minced with a sterile scalpel in a petri dish containing 5 mL of CO2-independent medium. Tissue pieces were then washed with 40 mL of FACS buffer (PBS supplemented with 2% fetal calf serum (FCS) and 2.5 mM EDTA) to recover single cell suspensions of thymocytes.  To generate scRNAseq data from M. domestica, 108 thymocytes were stained for 30 min at room temperature with a PE-labeled opossum MR1 tetramer loaded with 5-OP-RU, then washed and stained with 70 mL of anti-PE microbeads (Miltenyi).  Magnetic enrichment of tetramer-labeled thymocytes was then performed. Following magnetic enrichment, thymocytes were stained for 30 min at 4°C with the following primary antibodies: polyclonal rabbit anti-human CD3e (Dako A0452) and mouse anti-CD79A (clone HM57, GeneTex GTX74022) After washing with FACS buffer, opossum cells were further stained for 30 min on ice with the following secondary antibodies: donkey anti-rabbit IgG AF488 (Biolegend 406416) and anti-mouse IgG1 AF700 (clone RMG1-1, Biolegend 406631).  Cells were then washed with FACS buffer and stained with DAPI (Sigma). DAPI- Tetramer+ CD3+ thymocytes were flow-sorted (Aria III, BD) into PBS supplemented with 0.35% BSA (Sigma). Sorted cells were centrifuged, counted and 5,000 – 10,000 cells were loaded onto the Chromium 3' chip. Reverse transcription, library preparation and sequencing were performed according to manufacturer's recommendations (10x Genomics). Library was performed according to the manufacter's instructions (single cell 3' v3 protocol, 10x Genomics). Briefly, GCs were resuspended in the master mix and loaded together with partitioning oil and gel beads into the chip to generate the gel bead-in-emulsion (GEM). The poly-A RNA from the cell lysate contained in every single GEM was retrotranscripted to cDNA, which contains an Ilumina R1 primer sequence, Unique Molecular Identifier (UMI) and the 10x Barcode. The pooled barcoded cDNA was then cleaned up with Silane DynaBeads, amplified by PCR and the apropiated sized fragments were selected with SPRIselect reagent for subsequent library construction. During the library construction Ilumina R2 primer sequence, paired-end constructs with P5 and P7 sequences and a sample index were added.