GSM6001943: MB49-intra-3; Mus musculus; RNA-Seq
Tumor were dissected and frozen on dry ice, and the purification of total and small RNAs is prepared using the miRNeasy mini kit (Qiagen).  10-50 mg of frozen tissue samples, with the addition of 700 ul of Qiazol reagent, are  homogenized using Navy Rhino tubes in a Bullet Blender Homogenizer (Next Advance) for 5 minutes. After chloroform extraction, The sample is then applied to the miRNeasy Mini spin column. On-column DNAse digestion is also performed to remove any residual genomic DNA contamination. Quantitative assessment of the purified total RNA is then accomplished by using a Qubit Broad Range RNA kit (Thermofisher), and concentration is determined by Ribogreen fluorescent binding to isolated RNA. The sequencing libraries are prepared with the RNA HyperPrep Kit with RiboErase (HMR)kit (Roche Sequencing Solutions), from 500ng total RNA, following the manufacturer's instructions. The resulting pool is then loaded into the appropriate NovaSeq Reagent cartridge, as determined by the number of sequencing cycles desired, and sequenced on a NovaSeq6000 following the manufacturer's recommended protocol (Illumina Inc.).