GSM4176004: Gradient 2 Fraction 4; Mus musculus; RNA-Seq
Wild-type FVB female mouse livers were dissected and washed in ice cold PBS. Tissue was placed in a tube containing 0.25 M buffered sucrose solution, 20mM Tris, water supplemented with protease inhibitor cocktail and 10 mM ribonucleoside-vanadyl complex (VRC) as a ribonuclease inhibitor) with 2.8 mm ceramic beads and placed in a bead homogenizer to homogenize tissue. Homogenized tissue was centrifuged at 5000 x g for 10 minutes to remove nuclei. A Biocomp Gradient Master™ was using to generate an 11 mL 10-50% sucrose gradient (with 10 mM VRC). Homogenate was layered onto the gradient, and components were resolved by ultracentrifugation in an SW41 rotor for 3 hours at 30,000 rpm (4° C). Twenty-four 0.5 ml fractions were collected from the gradient using the BioComp Piston Gradient Fractionator™. Fractions were split for RNA and protein extraction. RNA was extracted from each fraction by Direct-zol RNA miniprep kit. 10 equivalents of EDTA (relative to the VRC concentration) were added to each sample in Trizol-reagent before ethanol was added to remove the ribonucleoside-vanadyl complex. Protein concentrations were measured by the Pierce BCA protein assay kit. RNA libraries were prepared for sequencing using the KAPA stranded RNA-Seq kit with RiboErase. Libraries were multiplexed and pooled.
GEO
GSM4176004
GEO Accession
GSM4176004