GSM8374227: TT-CITE-4-GEX; Homo sapiens; OTHER
Pediatric thymus from children undergoing cardiac surgery were obtained and used according to and with the approval of the Medical Ethical Commission of Ghent University Hospital (Belgium). Thymus tissue was cut into small pieces and digested with 1.6mg/ml collagenase (Gibco, 17104-019) in IMDM medium for 30min at 37°C to generate a single cell suspension. The reaction was quenched with 10% FBS and the thymocyte suspension was passed through a 70μm filter to remove undigested tissue. Cells were frozen in FBS containing 10% DMSO and stored in liquid nitrogen until needed. Upon thawing, thymocytes were purified with a bead-based dead cell removal kit (Miltenyi, 130-090-101). 2x10^6 cells were blocked with Human TruStain FcX Blocking solution (BioLegend, 422301) and then stained with TotalSeq-C antibodies (customised panel containing TotalSeq-C Human Universal Cocktail 1.0 (BioLegend, 399905) and 13 individual TotalSeq-C antibodies) and anti-CD3-PE. Cells were washed and sorted for CD3. Around 25.000 cells per sample were loaded per well on Chip K (10x Genomics, 1000286) and GEMs were generated using a Chromium Controller (10x Genomics). Feature barcode (FB), gene expression (GEX) and TCR libraries were prepared according to protocol CG000330 Rev A (10x Genomics) using the Chromium Next GEM Single Cell 5ʹ GEM Kit v2 (10x Genomics, 1000244), Library Construction Kit (10x Genomics, 1000190), 5' Feature Barcode Kit (10x Genomics, 1000256), Human TCR Amplification Kit (10x Genomics, 1000252), Dual Index Kit TT set A (10x Genomics, 1000215) and Dual Index Kit TN set A (10x Genomics, 1000250). The protocol version for >6000 cells was followed and libraries were amplified for 13 cycles (cDNA), 14 cycles (GEX), 8 cycles (FB) or 12+10 cycles (TCR libraries). Library quality and quantity were checked on a Bioanalyzer instrument (Agilent) using a High Sensitivity DNA Assay. Libraries were pooled and sequenced on a Novaseq6000 instrument (Illumina) to a minimum of 25.000 reads/cell for GEX, 10.000 reads/cell for FB, and 5000 reads/cell for TCR libraries.