Rab46 and the liver
Rab46 and the liver
Homozygous (Efcab4b-/-) and wild-type (Efcab4b+/+) WT mice were weighed at 8 weeks and 12 weeks where they were sacrificed.  Liver tissue was placed in a tube with a metal lysis bead with 1 ml Trizol Reagent.  Tubes were then placed in a tissue lyser and agitated at 26 Hz for 3 x 1 min sessions. The resultant liquid was transferred to 1.5 ml centrifuge tubes, 200 μl of phenol-chloroform were added and left to settle for 3 mins at room temperature, then spun at 12000g (4°C) for 15 mins. The supernatant (top aqueous phase) was transferred to new tubes and 500 μl of isopropanol were added to each tube and gently mixed before being left to stand for 10 mins. The samples were then spun for 15 mins at 12000g (4°C), and the supernatant discarded. The pellets were re-suspended in 1 ml of 75% ethanol (in dH2O) before a further spin at 8000g (4°C) for 5min. The supernatant was removed carefully, the pellet dried and then 20-50 μl of RNA-free dH2O was added to each sample which were then stored at -80°C. RNA was quantified using a NanoDrop®.  The library preparation was constructed using the TruSeq Stranded mRNA kit (Illumina), following the manufacturer's recommended protocol.