GSM6970253: Brain, Phe31KO, repA; Mus musculus; RNA-Seq
3 mm x 3 mm tissue pieces (liver or brain) were homogenized in 200 l of cell extraction buffer (CEB; 100 mM Tris, 2 mM Na3VO4, 100 mM NaCl, 1% Triton X-100, 1 mM EDTA, 10% glycerol, 1 mM EGTA, 0.1% SDS, 1 mM NaF, 0.5% deoxycholate, 20 mM Na4P2O7), pH 7.4, containing PhosSTOP Phosphatase Inhibitor Cocktail (Roche) and EDTA-free Complete protease inhibitor cocktail (Roche). The homogenates were prepared using a bead beater and the homogenates were centrifuged at 9,000 g for 5 min at 4C. The previous steps were repeated until a clear tissue homogenate was produced. The tissue homogenate protein concentration was quantified using the bicinchoninic acid (BCA) assay using BSA as a standard. The tissues were snap frozen and stored at -80oC prior to RNA isolation and RNA was isolated using the miRNeasy Mini kit (Qiagen) incorporating an on-column RNase-free DNase digestion to remove all DNA. RNA (2-5 g) was resolved on 1.2% agarose formaldehyde gels, then transferred to 0.45 m Hybond-N+ nitrocellulose membrane (GE Lifesciences) and hybridized with biotinylated oligonucleotide probes speci?c to nuclear tRNAs and 18S rRNA (probe sequences are listed in Table S4). Hybridizations were carried out overnight at 50C in 5x SSC, 20 mM Na2HPO4, 7% SDS and 100 g.ml-1 heparin, followed by washing. The signal was detected using streptavidin-linked infrared-labelled antibody (diluted 1: 10,000 in 3x SSC, 5% SDS, 25 mM Na2HPO4, pH 7.5) by the Odyssey Infrared Imaging System (LI-COR Biosciences). RNA sequencing librarys were constructed using the Illumina TruSeq Sample Prep Kit according to the manufacturers instructions and sequenced on an Illumina NovaSeq 6000.