| Construction protocol: |
Following resection in the operating room, samples from the tumour and adjacent lung tissue from the same resection specimen at maximal distance (>5 cm) from the tumour were isolated, and transported rapidly to the research facility. Upon arrival, samples were rinsed with PBS, and the tumour sample macroscopically examined for tumour positioning. The tumour sample was subsequently divided in 3 pieces, with one piece containing mainly tissue derived from the tumour core, one piece containing tissue mainly derived from the tumour edge, and a third piece originating from the position intermediate to both samples. Each sample was subsequently minced on ice to smaller pieces of less than 1 mm3, and transferred to 10 mL digestion medium. Samples were incubated for 15 minutes at 37°C, with manual shaking every 5 minutes. Samples were then vortexed for 10 seconds, and pipetted up and down for 1 minute using pipettes of descending sizes (25 mL, 10 mL and 5 mL). Next, 30 mL ice-cold phosphate-buffered saline, pH 7.4 (PBS; ThermoFisher Scientific) containing 2% foetal bovine serum (ThermoFisher Scientific) was added, samples were filtered using a 40µm nylon mesh (ThermoFisher Scientific). Following centrifugation at 120 G at 4°C for 5 minutes, the supernatant was decanted and discarded, and the cell pellet was resuspended in 2 mL red blood cell lysis buffer and transferred to a 2 mL DNA low bind tube. Following a 5-minute incubation at room temperature, samples were centrifuged (120 G, 4°C, 5 min) using a swing-out rotor. Samples were next resuspended in 1 mL PBS containing 8 µL UltraPure bovine serum albumin (50 mg/mL; AM2616, ThermoFisher Scientific) and filtered over Scienceware Flowmi 40 µm cell strainers (VWR) using wide bore 1 mL low retention filter tips (Mettler-Toledo). 10 µL of this cell suspension was next counted using an automated cell counter (Luna) to determine the concentration of live cells. Single cell suspensions were converted to barcoded Drop-seq libraries by using the Chromium Single Cell 3’ Library, Gel Bead & Multiplex Kit and Chip Kit (10X Genomics; Pleasanton, California, USA), aiming for an estimated 4,000 cells per library. Samples were processed using kits pertaining to either the V1 or V2 barcoding chemistry of 10X Genomics. |
