GSM8413553: LEC,KrasG12D, scRNAseq; Mus musculus; RNA-Seq
The lungs were perfused with Heparin/DPBS pH 7.2 solution to remove red blood cells. The lobes of the lung were isolated and cut into pieces with a scalpel before placing them into milteny C-tubes (Miltenyi Biotec; catalog # 130-093-237) containing 5 ml of the lung dissociation cocktail (Miltenyi Biotec; catalog # 130-095-927). The C-tubes containing the tissue/enzyme mix were then placed into the sleeve of the gentleMACS dissociator and the m_lung_01 program was run once to dissociate the cells. The C-tubes were then placed in a 37°C incubator under continuous agitation for 45 minutes. Following incubation, the C-tubes containing the tissue/enzyme mix were placed into the gentleMACS dissociator and the m_lung_02 program was run to dissociate the cells. The C-tubes were then centrifuged at 300xg to collect the sample at the bottom of the tube. The sample was resuspended with 1x Buffer S and applied to a prewet 100 µm cell strainer in a 50 ml conical tube. This process was repeated with a 70 µm and 40 µm cell strainer to ensure that all cells were in a single cell suspension. The cell suspension was transferred to a 15 ml conical tube and centrifuged at 300xg to collect a pellet. The pellet was resuspended in 3% FBS in 1XDPBS for subsequent FACS sorting and live GFP positive lymphatic endothelial cells were submited to the genomics core for sequencing. The 10x Genomics Chromium Next GEM Single Cell Reagent Kits v3.1 (Dual Index) was used according to the manufacturer's protocol (Chromium Next GEM Single Cell 3' Kit v3.1, 4 rxns PN-1000269). The 10X Genomics Chromium iX Single Cell Analysis System which utilizes the NEXT GEM technology was used for partitioning and barcoding cells. The Eppendorf Mastercycler X50S was used for PCR amplification for 12 PCR cycles. libraries were sequenced on the illumina NovaSeq6000 sequencer with S4 200 cycle flow cell PE-100.