GSM3044754: Patient T17 Malignant; Homo sapiens; RNA-Seq
Fresh human lung tumor samples were obtained from Stanford Tissue Bank. Tissues were cut into small pieces and dissociated into single cell suspensions by 45 minutes of collagenase I (STEMCELL Technologies) digestion. Dissociated single cells were suspended at 107 per mL in staining buffer (HBSS with 2% heat-inactivated calf serum). After blocking with 10 μg/mL rat IgG, cells were stained with the antibodies listed below. Stained cells were washed and re-suspended in staining buffer with 1 μg/mL DAPI, analyzed, and sorted with a FACS Aria II cell sorter (BD Biosciences). Antibodies used for experiments included CD45-A700 (pan-immune cell marker), CD31-PE (endothelial cell marker), EpCAM-APC (epithelial cell marker), CD10-PE-Cy7 (fibroblast marker). All antibodies were obtained from BioLegend. Total RNA was extracted from sorted cell populations (range: 500 to 25,000 cells). A median of ~30 ng total RNA was amplified using the Ovation RNA-Seq System V2 (Nugen). 1 mg of resulting cDNA was sheared by sonication (Covaris S2 System) to an average size of 150-200 bp and used to construct DNA libraries for sequencing using NEBNext DNA Library Prep Master Mix (New England Biolabs).  Finally, DNA libraries were sequenced in the Stanford Stem Cell Institute Genome Center on an Illumina HiSeq 2000.