GSM9065790: Invivo - IL27 Vector; Exvivo - Anti-PD-L1 Rep 2; Mus musculus; RNA-Seq
Preservation in RNAlater at 4oC lasted for up to 1 week, following manufacturer's protocols, to preserve RNA integrity for downstream applications. RNAlater was removed and 'dried' tumor tissues were kept in microcentrifuge tubes at -80°C. Total RNA was isolated from sections of these tumors. Briefly, 600 μL of RLT buffer + 10 μL of β-Mercaptoethanol were added to the tumor section, and homogenized with a PRO200 homogenizer (MidSci, ValleyPark, MO, USA) in three brief pulses of 10–15 s each at a mid-power. Lysates were then processed using a Qiagen RNAeasy kit (Qiagen). Total RNA was sequenced by LC Sciences (Houston, TX) with analysis support. Before sequencing, RNA integrity was checked with an Agilent 2100 Bioanalyzer to ensure a minimum RNA Integrity Number (RIN) of 8. Illumina's TruSeq-stranded-mRNA protocol was followed to prepare a poly (A) RNA sequencing library and Poly (A) tail-containing mRNAs were purified using oligo-dT magnetic beads. The purified poly (A) RNA was fragmented using divalent cation buffer in elevated temperature. Agilent Technologies 2100 Bioanalyzer High Sensitivity DNA Chip was used to perform quality control analysis and to quantify the sequencing library. Illumina's NovaSeq 6000 sequencing system was used to perform paired-ended sequencing.