title: GSM6172161: Lung cells mCherry-negative (1); Mus musculus; RNA-Seq
library_construction_protocol: Tumour bearing lungs from KrasG12D/+; p16FDR/+; Rosa26loxP-STOP-loxP-YFP/+ mice were dissected into 1 mm3 pieces and dissociated in a solution of 0.5% w/v collagenase type 2 (Lorne Laboratories Ltd.), 0.1x Trypsin (Gibco), 50 μg/ml DNaseI (Worthington) and 2.5 μg/ml fungizone (Gibco) in Hank's Balanced Salt Solution (HBSS (Gibco) for 45 minutes at 37°C. Tissue fragments were further dissociated into single cells through mechanical trituration using a pipette and filtration through a 70 μm filter. Single cells were washed in HBSS and suspended in FACS media (PBS supplemented with 1% foetal calf serum (PAA), 25 mM HEPES and 10 μg/ml of 4′,6-diamidino-2-phenylindole (DAPI, BD Pharmingen) for live/dead cell discrimination) before being analysed using a BD FACS AriaIII Flow Cytometer. Cells were collected into 4°C FACS media for 10X single cells RNA-sequencing analysis. Flow-sorted cells were centrifuged and resuspended in complete DMEM to a concentration of 700-1000 cells per microlitre. Cells were partitioned and barcoded using the 10X Genomics Chromium Controller and associated v3 kit components, according to manufacturer's instructions. Harvested cDNA was assessed and quantified using the Agilent 2100 Bioanalyser and library construction performed using the 10X Single Cell 3' Library Kit v3. Libraries were quality controlled by performing a MiSeq Nano PE100 run followed by deeper sequencing on the HiSeq2500 v4 system.