GSM1569641: Liver_8mkd-furan_FFPE18h_43_riboRNAseq; Mus musculus; RNA-Seq
Mice were anaesthetized under carbon dioxide inhalation followed by exsanguination. Livers were removed and divided into three: One piece was frozen, one piece was preserved in formalin for 18 hours, one piece was preserved in formalin for 3 weeks. FROZEN: samples were placed in cryovials and immediately snap frozen in liquid nitrogen, these tissues were stored at -80oC until RNA extraction was performed.  Total RNA was extracted from ~100mg frozen tissue using the RNeasy Midi Kit (Qiagen) according to the manufacturer's instructions.  Total RNA concentration and quality was determined using a Nanodrop 1000 Spectrophotometer and an Agilent 2100 Bioanalyzer, respectively.  Fresh-frozen RNA samples had A260/280 ratios > 1.83 and RINs > 8.9. FFPE: samples were preserved in formalin for 18 hours or 3 weeks and then stored in paraffin blocks at room temperature. RNA was extracted from 1x10uM section (18 hours samples) and 2x10uM sections (3 weeks samples) using the RNeasy FFPE RNA extraction kit (Qiagen). Total RNA concentration and quality was determined using a Nanodrop 1000 Spectrophotometer and an Agilent 2100 Bioanalyzer, respectively.  FFPE RNA samples had A260/280 ratios > 1.74 and RINs 1.3-2.4. Libraries were prepared according to Illumina's instructions accompanying the TruSeq Total RNA Sample Kit (Part# RS-122-2201). Briefly, RNA was depleted of ribosomal RNA via beads, fragmented, and converted into ds-cDNA.   The resultant cDNAs were barcoded and ligated to Illumina adapters.  After adapter ligation, the cDNA was PCR amplified. The purified cDNA was captured on an Illumina flow cell for cluster generation. Libraries were sequenced on the HiSeq 2000 following the manufacturer's protocols.