2025-04-13
ENA first public
ERC000011
ENA-CHECKLIST
Centro Nacional de Investigaciones Cardiovasculares Carlos III
INSDC center name
public
INSDC status
ERS23919700
SRA accession
E-MTAB-14985:N4_WT_day5
Submitter Id
ArrayExpress
broker name
Neonatal Cardiomyocyte
cell type
not collected
collection date
house mouse
common name
Protocols: 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
description
wild type genotype
genotype
not collected
geographic location (country and/or sea)
not applicable
isolate
Mus musculus
organism
Mus musculus
organism
Mus musculus
scientific_name
N4_WT_day5
title