GSM5746437: WT 2; Mus musculus; RNA-Seq
Mice were anesthetized using 2% isoflurane and subsequently sacrificed using cervical dislocation. The left lobe of the liver was collected from three mice per group and snap-frozen in liquid nitrogen. ~20 mg per sample was homogenized using a TissueLyser II (Qiagen) and total RNA using the Qiagen RNeasy Mini Kit (#74104). The RNA yield was determined using a High-sensitivity RNA ScreenTape Analysis (Agilent) on an Agilent TapeStation Instrument. This analysis was done by the Center for Cancer Research (CCR) Genomics Core. Samples with a RIN > 8.0 were sent for bulk mRNA sequencing. Sequencing was done by the CCR Sequencing Facility at the Frederick National Laboratory for Cancer Research. Briefly, the TruSeq mRNA library kit (Illumina) was used to prepare the mRNA library following the manufacturer's instructions. 200ng of total RNA was used as the input to an mRNA capture with oligo-dT coated magnetic beads. The mRNA was fragmented, and then a random-primed cDNA synthesis was performed. To prepare the library, the resulting double-strand cDNA was used as the input to a standard Illumina library prep with end-repair, adapter ligation and PCR amplification being performed. The final purified product was then quantitated by qPCR before cluster generation and sequencing. Six mRNA-Seq samples were pooled and sequenced on NextSeq using paired-end sequencing and a read length of 76 (2 x 76 cycles).