title: GSM6466253: pool_A_Single_Cell; Mus musculus; RNA-Seq
library_construction_protocol: Animals were anaesthetized with ketamine (100 mg/kg) and xylazine (10 mg/kg) (i.p.) and given 10 mU/g heparin sodium (i.p.) (LEO Pharma Inc., Thornhill, ON, Canada). The inferior vena cava was exposed surgically and the animals were bled and then 25 U/mL heparin in 10 mLs of sterile 0.9% saline was flushed through lungs via pulmonary artery until they were cleared of all evidence of red blood cells. Lungs were rinsed in PBS, cut into smaller pieces and placed into gentleMACS C tubes TM (Miltenyi Biotech, Bergisch Gladbach, Germany) containing 2.5 mLs DPBS to which 2.5 mLs of digestion enzyme mix were added. This mix included 2500 U Collagenase I, 30 U Neutral Protease/Dispase (Wothington Biochem., Lakewood, NJ, USA), and 500 U Deoxyribonuclease (Sigma-Aldrich, Oakville, ON, Canada) in 1x Dulbecco's PBS (Thermofischer Scientific, Burlington, ON, Canada), and was made fresh for each experiment and kept on ice. GentleMACS C tubes were placed into a temperature regulated gentleMACS Octo Dissociator TM (Miltenyi Biotech, Bergisch Gladbach, Germany) and underwent mechanical dissociation according to a custom mouse lung program at 37 ºC for 30 minutes. Dissociated tissue was passed through a pre-wetted 75 μm filter (Thermofischer Scientific, Burlington, ON, Canada), re-suspended with additional 5 mLs PBS and treated with 200 μLs 0.5M EDTA (Thermofischer Scientific, Burlington, ON, Canada). Pelleted cells were re-suspended in RBC-lysis buffer (Thermofischer Scientific, Burlington, ON, Canada) for 3 minutes at room temperature. Final cell pellet was re-suspended in 5 mLs PBS and cell counts and viability were performed using a Countess TM automated counter (Thermofischer Scientific, Burlington, ON, Canada). Barcoding of individual biological samples was performed as described by McGinnis et.al.(2019). Dissociated lung cells (0.5e+6 cells per sample) were suspended in 150 μLs solution containing a 1:1 molar ratio (200nM) of anchor and barcode oligonucleotide containing a unique sequence for each of the 12 samples to be processed. Samples were incubated for 13 minutes at room temperature with gentle mixing every 3-5 minutes. Next, a co-anchor (200nM) was added to stabilize the barcodes within the cell membrane and incubated for additional 5 minutes. Cells were washed twice in PBS,  cell counts were measured using a Countess(TM) automated counter (Thermofischer Scientific, Burlington, ON, Canada), and viability was measured based on the ratio of cells staining with trypan blue (Thermofischer Scientific, Burlington, ON, Canada). An equal number of cells from each 12 barcodes was pooled and 1000 cells/μL were further processed through the 10x-Genomics pipeline. Only samples with viability > 85% were used. Three libraries were constructed from the twelve pooled samples using the 10X Genomics Single Cell 3' RNA sequencing kit v3.