RNASeq of mice livers with different genotypes
RNASeq of mice livers with different genotypes
Following the three-week feeding period, the mice were euthanised by CO₂ followed by cardiac incision for serum preparation. The livers were excised, weighed and washed in PBS. The liver lobes were divided and each was flash-frozen in liquid nitrogen in a 1.5 ml reaction tube and stored at -80°C. To isolate total RNA from the mouse liver tissues, 200 µl of TRI Reagent® (Zymo Research Europe GmbH, Freiburg, Germany, catalogue no.: R2053) was dispensed into a 1.5 ml reaction tube. The liver tissues stored at -80°C were then placed on dry ice for the following procedure. A piece weighing approximately 5 mg was cut off with a scalpel and added to the TRI Reagent®. This was homogenised using the Mixy Professional tissue homogeniser (FastGene® Nippon Genetics, Düren, Germany), a further 200 µl of TRI Reagent® was added, the mixture was vortexed for 10 seconds and centrifuged for 1 minute at room temperature at 14.0 x g. The supernatant was transferred to a fresh 1.5 ml reaction tube and the RNA was then isolated using the kit in accordance with the manufacturer's instructions. The elution volume was 50 µl. Ribisomal RNA was depeleted using the NEB rRNA depeltion Kit and libraries for Illumina were preared using teh NEB NExt Ultra Express RNA Library Kit with 100ng input RNA 