GSM5573041: RNAseq_AEC2_2; Mus musculus; RNA-Seq
Mice were anaesthetised using Avertin. Skin and muscular layers were cut to expose the abdominal and thoracic cavities. Descending aorta was cut to exsanguinate and euthanise the animal. Cold PBS was perfused through the cardiac right ventricle to flush the blood out of the lungs. Lungs were then removed and inflated with 4% PFA and fixed overnight at 40C in PFA for histology. For isolation of live primary cells, lungs were instead inflated with lung/tumour dissociation buffer (Miltenyi Biotec), cut into small pieces and incubated at 370C for 30 minutes (normal lung) or 45 minutes (lung with neoplastic growth). A single cell suspension was obtained by mechanically dissociating using gentleMACS (Miltenyi Biotec) dissociator and passing the samples through a 70 µm filter. RBCs were lysed by resuspending the cells in ACK lysis buffer and leucocytes were depleted by using anti-CD45 microbeads (Miltenyi Biotec). Primary cells were isolated by FACS using SONY SH800 sorter. Sytox dyes (Blue, Green, Red and AADvanced) were used to exclude dead cells. Total RNA was isolated from primary cells using RNeasy Mini kit (Qiagen). Samples with RIN > 9 were used for library preparation by Duke University's genomics core using KAPA stranded mRNA library prep kit. Libraries were sequenced using Illumia Hi-Seq 4000 (50 bp single end).