GSM6215425: Gut, WT, 22months, rep3; Danio rerio; RNA-Seq
Whole tissues were dissected in cold PBS (Sigma-Aldrich), transferred to a microcentrifuge tube containing 100 μl of Trizol (Thermo Fisher Scientific), snap-frozen in dry ice and stored at -80°. To isolate the RNA, extra 50 μl of Trizol was added to each sample, and the tissue was homogenized with a mechanical homogenizer (VWR International) and a 1.5 pestle (Kimble Chase, Vineland, NJ, USA). After 5 min incubation at room temperature (RT), 30 μl of chloroform (1:5, VWR International) was added and the samples were incubated for further 3 min at RT before centrifuged at 13,000g, for 30 min, at 4°C. Isopropanol (Thermo Fisher Scientific) was then added to the aqueous phase of the solution, and the resultant mix was incubated for 10 min at RT, before centrifuged (13,000g for 15 min at 4°C). Finally, the pellet was twice washed in 250 μl of ice cold 75% ethanol and left to air-dry, before resuspended in 14 μl of nuclease-free water. RNA integrity was assessed with the bioanalyzer Agilent 2100 Bioanalyzer (Agilent, Santa Clara, CA, USA). All the samples had a RNA integrity number (RIN) ≥9. Library preparation was performed following the Illumina methodology. mRNA was extracted from 500 ng of RNA with oligo-dT beads, capturing poly(A) tails, using the NEBNext Poly(A) mRNA Magnetic Isolation Module (New England Biolabs Inc). cDNA libraries were made with the NEBNext® Ultra™ RNA Library Prep Kit for Illumina, following the manufacturer's instructions (New England Biolabs Inc).