GSM5964232: tumor, control; Mus musculus; RNA-Seq
30-60 mg tumor tissues were used for RNA extraction. A mass of stainless-steel beads (Next Advance, cat# SSB14B) equal to the mass of the tissue was added to the tube. Two volumes of TRI Reagent® (Zymo Research) were added to the tube, and samples were immediately mixed in the Bullet Blender (Next Advance) for 1 minute at a speed of ten. Samples were visually inspected to confirm desired homogenization and then incubated at 37 °C for 5 minutes. The TRI Reagent® was added up to 0.6 mL, and samples were mixed in the Bullet Blender for 1 minute. Total RNA was extracted using Direct-zol™ RNA Miniprep Kit (Zymo Research). Optical density values of extracted RNA were measured using a NanoDrop (Thermo Scientific) to confirm an A260:A280 ratio above 1.9. RNA integration number (RIN) was measured using BioAnalyzer (Agilent Technologies) RNA 6000 Nano Kit. The cDNA libraries were prepared using the QuantSeq 3'mRNA-Seq Library Prep Kit FWD for Illumina (Lexogen) as per the manufacturer's instructions. Briefly, total RNA was reverse transcribed using oligo (dT) primers. The second cDNA strand was synthesized by random priming, in a manner that DNA polymerase is efficiently stopped when reaching the next hybridized random primer, so only the fragment close to the 3' end gets captured for later indexed adapter ligation and PCR amplification. The processed libraries were assessed for their size distribution and concentration using BioAnalyzer High Sensitivity DNA Kit (Agilent Technologies). The denatured libraries were loaded onto an MiSeq v3 flow cell on an Illumina MiSeq and run for 151 cycles using a single-read recipe according to the manufacturer's instructions.