2025-04-15
ENA first public
ERC000011
ENA-CHECKLIST
Centro Nacional de Investigaciones Cardiovasculares (CNIC)
INSDC center name
public
INSDC status
ERS23862059
SRA accession
E-MTAB-14976:Sample 3_KI-
Submitter Id
16.5
week
age
ArrayExpress
broker name
not collected
collection date
house mouse
common name
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 intermediate region was cryopreserved in liquid nitrogen for subsequent extraction of nuclei. To isolate cell nuclei, we followed the protocol described in (81). Briefly, tissues from three animals per group were resuspended in lysis buffer and homogenized using a Dounce homogenizer and a T10 UltraTurrax probe homogenizer. The homogenate was then filtered through a series of cell strainers (subsequent passages on 100, 70 and 40 um strainers), and centrifuged at 1,000 g for 10 minutes to pellet the nuclei, which were washed twice with nuclei storage buffer (2.5M Sucrose, 1M Tris-HCl, pH = 7.2, 1M KCl, 1M MgCl2, 2M spermidine, protease inhibitor cocktail set III-EDTA-free, 40 units/mL RNase OUT) and resuspended in 2% BSA for subsequent purification by cell sorting. We enriched the sample in diploid and tetraploid nuclei based on their DAPI staining intensity in a FACSAria cell sorter (BD Biosciences). Purified nuclei were then processed for sequencing by CNIC’s Genomics Unit using 10X Genomics platform. 10X Genomics protocol was followed by CNIC’s Genomics Unit using 10X Genomics platform.
description
adult
developmental stage
TEVs-TTN
genotype
not collected
geographic location (country and/or sea)
Pool: DUT1782 DUT1885 DUT1886
individual
not applicable
isolate
Mus musculus
organism
Mus musculus
organism
heart
organism part
Mus musculus
scientific_name
male
sex
C57Bl6/j
strain
Sample 3_KI-
title