GSM7567334: CD3KO_MR1KO_chimera; Mus musculus; OTHER
Single-cell suspension of thymus was obtained as described (Salou et al. 2019). MR1:5-OP-RU tetramer staining along with an anti-TCRb-APC (clone H57-597; 1:200) was performed at room temperature for 30min in PBS 0.5% BSA 2 mM EDTA complemented with rat anti-mouse CD16/CD32 antibody and MR1:6FP to block non-specific binding following the National Institutes of Health (NIH) Tetramer Core Facility instructions. Tetramer-based magnetic enrichment was performed as described (F. P. Legoux and Moon 2012). Cells were washed and stained for surface markers with following monoclonal antibodies: anti-CD44-APC-Cy7 (IM7, Biolegend; 1:400), -CD24-PE-Cy5 (M1/69, Biolegend; 1:1600), -CD19-AF700 (ID3, Biolegend; 1:800), -B220-AF700 (RA3-6B2, eBioscience; 1:400), -CD11c-AF700 (N418, eBioscience; 1:200), -F4/80-AF700 (BM8, Biolegend; 1:200). For cell hashing in combination with 10x Chromium Single Cell 5′ Solution, TotalSeq-C anti-mouse Hashtags 2 to 7 (M1/42; 39-F11, Biolegend, 155863, 155865, 155867, 155869, 155871, 155873; 1:100 for all) were used according to manufacturer recommendations. Single-cell suspensions from 6 hematopoietic chimeras thymi were pooled and DAPI (1:1000) staining was added just before FACS sorting on an Aria III cell sorter (BD). Total TCRb+MR1:5-OP-RU+ cells were flow-sorted into PBS 0.35% BSA, centrifuged, counted, and 6,500 cells were loaded onto the Chromium 5′ chip. Reverse transcription, library preparation and sequencing were performed according to manufacturer recommendations (10× Genomics). Three libraries were performed according to the manufacter's instructions (single cell 5' with feature barcode v2 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). Total 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. 5' VDJ scRNA-seq CITE-seq