title: GSM6700930: scRNASeq,Trp53_KO,Rep1; Mus musculus; RNA-Seq
library_construction_protocol: Ascites fluid was immediately transferred to 10ml EDTA-coated tubes that were centrifuged at 600xg for 10 minutes at 4°C in a swing bucket rotor centrifuge. Cleared ascites fluid was discarded, and cell pellet was resuspended in 5ml 1x ACK buffer (150mM NH4Cl, 10mM KHCO3, 0.1 mM Na2EDTA) to lyse red blood cells. Following 1 minute incubation, cells were centrifuged 350xg for 5 minutes at 4°C. Supernatant containing lysed red blood cells was discarded, and 1x ACK treatment followed by centrifugation was repeated once more. Remaining cells were washed with 1x PBS to remove ACK buffer.Cells were then washed on 0.2ml FACS buffer (2% FBS or 0.5% BSA in 1xPBS). To identify viable cells, cells were stained with LIVE/DEAD® Fixable Violet Dead Cell Dye (ThermoFisher Scientific, catalog No. L-34963). Extracellular surface markers (CD45) were subsequently stained at 4°C for 30 minutes directly in polypropylene tubes. Antibody dilutions were typically 1:100 in FACS buffer. Cells were centrifuged for 5 minutes at 300-400xg, and supernatant containing antibodies was discarded. Live cells were immediately subjected to FACS by the SickKids-UHN Flow Cytometry Facility. At least sorted 4000 cells / group were then immediately subjected to 10x Genomics library preparation by the Princess Margaret Cancer Genomics Centre for sequencing on an Illumina HiSeq2500. At least 4000 sorted cells / group were subjected to 10x Genomics library preparation as per manufacturer's protocol by the Princess Margaret Cancer Genomics Centre for sequencing on an Illumina HiSeq2500.