RNAseq of titin cleavage in neonatal mouse cardiomyocytes
RNAseq of titin cleavage in neonatal mouse cardiomyocytes
Neonatal mouse cardiomyocytes were isolated from 6-8 pups per experimental group. Dissected hearts from one-to-three-day-old mice were minced with a scissor in cold Hanks' Balanced Salt Solution (HBSS) and dissociated using the Pierce Primary Cardiomyocyte Isolation Kit (Thermo ScientificTMPierceTM, 88281) in 0.21 mL enzyme mix/heart in a 2 mL reaction tube for 20 minutes at 37°C. After centrifugation, the pellet was washed twice with HBSS and resuspended in 0.5 mL cardiomyocyte medium (DMEM for Primary Cell Isolation containing 10% heat-inactivated FBS and 1% penicillin/streptomycin)/heart and plated onto MatTek dishes covered with matrigel solution (Fisher Scientific, 11553620). 24-48h after plating, cardiomyocytes were infected with AAV6-expressing GFP-TEVp (AAV6-TEVp) at 13,450 multiplicity of infection. Media were replaced with cardiomyocyte medium 16 h after virus addition. RNA was isolated from neonatal cardiomyocytes cultured on 24-well plates (Mattek, P24G-1.5-13-F), before infection (WT_day0; Halo_day0) or -2, -5 days post infection with AAV6-TEVp (WT_day2; WT_day5; Halo_day2; Halo_day5). 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