title: wildtype, heart, 5 months post fertilization, replicate 1
design: Tissue collection and dissection: Adult zebrafish were euthanized by exposure to chilled water (0-4C). Gills, heart and kidney were collected from 32 individual fish at two time-points, namely at 3 and 5 mpf for klotho mutants and wildtype sibling controls (AB background; 8 fish per genotype per time-point - 4 males, 4 females) for a total of 96 samples. Tissues were dissected in cold PBS and immediately stored in RNALater Stabilization Solution (ThermoFisher Cat# AM7021). RNALater was removed after an overnight incubation at 4C, and samples were stored at -80C until processing. RNA extraction: All tissues were homogenized using the TissueLyser II (Qiagen) plus Lysis buffer containing -mercaptoethanol and stored at -80C. RNA extraction was performed using the automated protocol in the QIAcube workstation utilizing the RNeasy Fibrous Tissue Mini kit (Qiagen Cat No./ID: 74704). RNA integrity and quality were assessed by Agilent TapeStation using High Sensitivity RNA ScreenTapes. Samples were normalized and 300ng of RNA was used for library prep for each sample. ERCC RNA Spike-in mix was added for quality control. mRNA was captured using poly-T oligos attached beads for library selection.