GSM8684969: p29_TEC; Homo sapiens; RNA-Seq
Thymus or thymoma samples were cut into 1 mm3 pieces using a scalpel. Tissues were digested enzymatically for 20-40 min at 37°C under agitation, in CO2-independent medium (Invitrogen) containing collagenase I (2 mg/ml; Sigma-Aldrich), hyaluronidase (2 mg/ml; SigmaAldrich), and deoxyribonuclease (25 mg/ml; Sigma-Aldrich). The tissue pieces were then dissociated with a 20ml syringe plunger on a 40 µm cell strainer (BD) in 1 % Fetal Calf Serum (FCS), 2 mM EDTA phosphate-buffered saline (PBS) (Invitrogen) until uniform cell suspensions were obtained. The suspended cells were subsequently centrifuged for 10 min at 400 g before either staining for flow cytometry, or CD3+ T cell enrichment followed by scRNA-seq as described below.  Thymic tissue samples were cut into ~1 cm3 pieces in PBS. The bulk of thymocytes was removed by a gentle pressure on the tissue fragment. After removing the supernatant, the remaining tissues was cut into fragments of 2-4 mm. Three sequential digestion steps were then performed. 1) 10 mL Collagenase A (2 mg/ml)/DNAseI (0.1 mg/mL) in 10 mL RPMI for 45 min at 37°C under slow rotation (190 rpm). Centrifugation at 110 g for 2 min was then performed to sediment fragment and supernatant was discarded. 2) a digestion, identic to the first one, was performed on the remaining tissue. 3) tissue remnants were incubated in 0.05% Trypsin in the collagenase/DNAse mix solution for 45min at 37° with constant agitation (190 rpm). During the last 10-15 min of the incubation, FCS (1:5) was added to neutralize Trypsin activity. After centrifugation (110 g for 2 min), the supernatant was collected before sorting the TECs. Alive CD45-EPCAM+ cells were sorted using a HP800 SONY sorter before scRNA-analysis T cells were loaded into a Chromium Single Cell 5′ V(D)J Enrichment Kit, Human T Cell (10X Genomics), according to the manufacturer's instructions. The thymoma TEC were analyzed using scRNAseq (3' chromium kit) 10x genomics.