GSM5293752: MY-R25; Mus musculus; RNA-Seq
Primary tumors were dissociated using collagenase IV, dispase, and trypsin at 37 °C for 30 min. After dissociation, the samples remained continually on ice, were in contact with ice-cold solutions, and were in the presence of 2 mM EDTA and 1 U/ml DNase to prevent aggregation. Cells were stained with antibodies to 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 All Prep kit (Qiagen, 80284). RNA quality of each tumor sample was assessed using the RNA6000 PicoAssay for the Agilent 2100 Bioanalyzer as per the manufacturer's recommendation. 4.4 ng total RNA per sample was used for cDNA synthesis and library preparation using Trio RNA-Seq, Mouse rRNA kit (Tecan, 0507-32), according to the manufacturer's instructions. The purified cDNA library products were evaluated using the Agilent bioanalyzer and sequenced on NextSeq High Output 1x75 (Admera Health Biopharma Services).