organism: Mus musculus
description: Protocols: Mice were housed in ventilated cages with free access to food and water and 12h day/night cycles. Heterozygous TEVs-TTN animals were crossed to obtain WT and homozygous animals. TEVs-TTN animals were described before, and 5´-CGTGGTGGCTTATCTTCTAGC-3´ and 5´-CTGTTGGTTCATGCATCTCC-3´ primers were used for genotyping. Only male mice were used for the experiments. Animals were handled in accordance with European and Spanish guidelines for animal welfare and with the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health. The protocol was approved by the Ethics Committee of Animal Experiments of our institutional committees (project numbers PROEX 042/18 and PROEX 107.8/23). WT and homozygous TEVs-TTN mice aged 8 - 26 weeks were injected with AAVMYO viral particles or 100 µl PBS, 0.001% pluronic acid via the tail vein. Mice were then returned to their breeding cages until sacrifice by CO2 inhalation six days post infection (dpi). Upon sacrifice, the heart together with the ascending aorta and pulmonary trunk, were dissected out. The ascending aorta, from the sinotubular junction to the branching of the brachiocephalic artery and atrial myocardium were excised and discarded. Transverse ventricular samples were obtained from which the most apical region was cryopreserved for protein and RNA extraction. RNA was isolated from hearts stored at -80ºC in 500 μL of RNAlater™ Stabilization Solution (Invitrogen, Cat. No. AM7020) homogenized with T10 basic ULTRA-TURRAX® (IKA) or from neonatal cardiomyocytes cultured on 24-well plates (Mattek, P24G-1.5-13-F). Total RNA was isolated by using Trizol (ThermoFisher Scientific) followed by chloroform precipitation and a purification step using RNeasy micro-Kit (Qiagen, Cat. No. 74004) following the indications of the provider. RNA quantity was quantified from its absorbance at 260 nm, while purity was assessed from the ratios of absorbance at 260 nm and 280 nm, and between 260 nm and 230 nm. Sample quality was evaluated using RNA Integrity Number (RIN), which was measured using a 6000 Nano chip in a 2100 Bioanalyzer (Agilent Technologies). For next generation sequencing (NGS) experiments, only RNA samples with RIN > 9 were used. Messenger RNA was purified from total RNA using poly-T oligo-attached magnetic beads. After fragmentation, the first strand cDNA was synthesized using random hexamer primers followed by second strand cDNA synthesis. The library was ready after end repair, A-tailing, adapter ligation, size selection, amplification, and purification.  The kit for library preparation was the Novogene NGS RNA Library Prep Set (PT042). The library was checked with Qubit 2.0 and real-time PCR for quantification, and a bioanalyzer 2100 for size distribution detection.