GSM6742868: aP2-Cre tdTomato+ 3; Mus musculus; RNA-Seq
For tumors, tumor samples were manually dissected from normal surrounding tissue. For sorted cells, tissues were manually dissociated and then digested for 1 hour at 37°C in 2 U/mL Collagenase B (#11088831001, Roche)/Dispase II (#04942078001, Roche), 50 mM HEPES/KOH pH 7.4; 150 mM NaCl. Cells were then spun down and resuspended in 2X volume 10% fetal bovine serum (FBS) (#SH30910.03, GE Hyclone) in phosphate buffered saline (PBS) to inactive digestion enzymes, samples were sequentially filtered through 70 μm (#22363548, Fisher) and 40 μm (#22363547, Fisher) filters to yield single cell suspensions. Data was acquired on a FACS Aria Cellsorter (BD Biosciences) and analyzed using FACSDiva software. DAPI used as a live/dead cell marker. RNA from tumors was harvested using miRNeasy mini plus kit (Qiagen) and RNA from sorted cells was harvested using miRNeasy micro kit (Qiagen). RNA libraries were prepared for sequencing using standard Illumina protocols at the St. Jude Hartwell Center.