GSM5419443: rSP52; Mus musculus; RNA-Seq
Microdissected lung tumors were dissociated with collagenase IV, dispase, and trypsin at 37 °C for 30 min. After dissociation, the samples were maintained on ice, in the presence of 2 mM EDTA and 1 U/mL DNase to prevent aggregation. For the sorting of neoplastic cells, the dissociated cells were stained with antibodies against CD45 (BioLegened, 103112), CD31 (BioLegend, 303116), F4/80 (BioLegend, 123116), and Ter119 (BioLegend, 116212) to exclude hematopoietic and endothelial cells (lineage-positive (Lin+) cells). DAPI was used to exclude dead cells. FACSAria sorters (BD Biosciences) were used for cell sorting.RNA was purified using RNA/DNA Allprep kit (Qiagen, 80284). RNA quality was assessed using the RNA6000 PicoAssay for the Agilent 2100 Bioanalyzer. Two to ten nanograms of total RNA served as input for the preparation of RNA-Seq libraries using the Trio RNA-Seq, Mouse rRNA kit (Tecan Genomics: 0507-32). Libraries were constructed per the manufacturer's instructions. Purified libraries were assessed using the Agilent High Sensitivity DNA kit (Agilent Technologies: 5067-4626).