GSM9249370: Exercise mouse 5; Mus musculus; RNA-Seq
The specimens used for RNA isolation were taken from the mice's whole heart consisting of both atriums (including the left and right atrial appendages) and ventricles. The animal's hearts were soaked directly after extraction into Trizol® (Life Technologies, Carlsbad, USA) (1 ml Trizol® reagent was used pro 100 mg heart tissue) and homogenized using IKA T10 Ultra-Turrax basic Homogenizer (Janke & Kunkel KG, Staufen i. Breisgau). The chloroform solution (Sigma-Aldrich, Steinheim, Germany) was added to the homogenized tissues with 0.2 ml pro milliliter Trizol. The mixture was agitated for 15 seconds and incubated at room temperature for 2-3 minutes. The samples were centrifuged 13000 rpm at 4oC for 15 minutes to separate the upper aqueous-, lower emulsion- and middle interphase. The upper aqueous phase containing the desired RNA was transferred into new clean tubes. The isolated RNA was washed by agitation for 15 seconds with 500 µl Isopropanol 99.7% (Sigma-Aldrich, Steinheim, Germany), each 1 ml sample followed by 10 minutes incubation at room temperature. Subsequently, the samples were recentrifuged with the same settings as previously described. The pellets containing the precipitated RNA were afterward cleansed with 1 ml 75% Ethanol followed by centrifugation at 10000 rpm at 4oC for 8 minutes. The supernatant was discharged, and the isolated pellet containing the desired RNA was dried at room temperature before adding 30 µl RNA-free water for dilution. NEBNext® Ultra II Directional RNA Library Prep technology