GSM8703029: Alt-1a; Mus musculus; RNA-Seq
Upon sacrifice, tumors were surgically removed from the mice, and small pieces of the tumors were cut and snap-frozen in liquid nitrogen. RNA was isolated from snap-frozen tumor sections using a combination of TRIZOL (#15596026, Thermo Fisher Scientific) and RNeasy micro kits (#74004, Qiagen, Hilden, Germany). In brief, tumor sections were combined with 5 mm stainless steel beads (69989, Qiagen) in 1 mL of ice-cold TRIZOL into 2 mL microcentrifuge tubes. The samples were then homogenized by placing the tubes in a TissueLyser II device (#85300, Qiagen) set to shake at 30 Hz for 2 minutes. After ensuring samples were completely homogenized, the lysate was transferred to a new 1.5 mL tube without the stainless steel beads. Samples were allowed to incubate at room temperature in TRIZOL for 5 minutes. 0.2 mL of chloroform was added to each sample, shaken vigorously, and allowed to incubate at room temperature for 2-3 minutes. The samples were centrifuged at 4 for 30 minutes at 12,000 xg. The aqueous top layer was carefully removed and added to a new tube containing 0.5 mL 70% ethanol and mixed carefully by pipetting up and down several times. The RNA and ethanol mix was applied to the RNeasy spin columns via centrifugation at 10,000 xg for 30 seconds. RNA and ethanol mix was applied to the columns in 0.5 mL aliquots until all samples had been applied to the column. The samples were washed with 0.7 mL RW1 buffer and centrifuged at 10,000 xg for 30 seconds. The flow-through was discarded, and samples were washed with 0.5 mL RPE buffer and centrifuged at 10,000 xg for 30 seconds. The flow-through was discarded, and samples were washed with 0.5 mL RPE buffer and centrifuged at 10,000 xg for 2 minutes. The flow through was discarded, and the columns were placed into fresh collection tubes and centrifuged at 10,000 xg for an additional 1 minute. Then RNA was eluted in 35 µLof RNase-free water and centrifuged at max speed (14,000 xg) for 1 minute in fresh 1.5 mL microcentrifuge tubes. RNA concentration and purity were measured via nanodrop. Samples were stored at -80 until further use. RNA samples were submitted to the Vanderbilt Technologies for Advanced Genomics (VANTAGE) core facility for bulk RNA sequencing. Samples underwent quality control via RNA ScreenTape to ensure all samples had a RINe greater than 7. All of our samples had RINe numbers greater than 8.1, and most were greater than 9.0. The VANTAGE core performed mRNA enrichment and cDNA library preparation via the stranded mRNA Library Prep kit (New England Biolabs, Ipswich, MA, USA). Paired-End 150 bp sequencing was performed on the Illumina NovaSeq 6000 targeting an average of 50M reads per sample.