GSM8449885: KRAS wild type AT2 cells run 1; Mus musculus; RNA-Seq
Four weeks postinfection with Ad5-CMV-Cre mice with the genotype KRAS(+/LSLG12V):ROSA26(LSLeYFP/LSLeYFP) and their KRAS wild type littermates were anesthetized and lungs were perfused. Dispase I was instilled into the lungs through tracheal intubation. 1% certified low melt agarose was also instilled. Lungs were introduced in HBSS and incubated at room temperature for 45 min. After this time, lungs were minced, incubated for 4 min in DMEM containing 0.1 mg/ml DNase I and 25mM Hepes, and passed through 100 µm and 40 µm cell strainers. After removing red blood cells with the ACK Lysing Buffer cells were stained for subsequent Fluorescence-activated Cell Sorting (FACS). Non-endothelial cells (CD31—) and non-leukocytes cells (CD45—), epithelial cells (EpCAM-PEmed) and positive for MHC-II (I-A/I-E)-PE-Cy7 were considered AT2 cells. Single cell lysis, reverse transcription and preamplification were performed overnight on the C1™ system. The following day, samples were harvested and libraries for single-cell mRNA next generation sequencing were prepared using the Illumina Nextera XT DNA Library preparation kit according to Fluidigm's instructions.