2025-06-02
ENA first public
ERC000011
ENA-CHECKLIST
University Hospital Heidelberg Department of Cardiology
INSDC center name
public
INSDC status
ERS21395781
SRA accession
E-MTAB-14573:WT_LFD_#436
Submitter Id
20
week
age
ArrayExpress
broker name
not collected
collection date
house mouse
common name
Protocols: CM nuclei were purified from each flash frozen left ventricle of experimental mice of 4 groups (n=3-4 per group) using fluorescence-activated sorting. Each heart piece was homogenized using the gentleMACS dissociator (Miltenyi) in an MTube with 2ml lysis buffer (5mM CaCl2, 3mM MgAc, 2mM EDTA, 0.5mM EGTA, 10mM Tris-HCl, pH 8) on the ‘protein_01’ mode. 2ml lysis buffer containing 0.4% NP40 were added to the crude homogenate, filtered and centrifuged (1000g, 5min, 4°C). The nuclei pellet was then resuspended in staining buffer (1% BSA, 0.1% Tween and 0,05% (w/v) Glycin in PBS) containing rabbit anti-PCM-1 antibody (1:1000, HPA023374, Sigma-Aldrich) and mouse anti-PLN antibody (1:1000, A010-14, Badrilla) for 20 minutes at 4°C. Anti-rabbit secondary antibody conjugated to Alexa 568 (1:1000, A11011, Life Technologies) and anti-mouse secondary antibody conjugated to Alexa 488 (1:1000, A11029, Life Technologies) were then added and incubated for 20 additional minutes. After centrifugation (1000g, 5min, 4°C) the nuclei were resuspended in PBS with 1%BSA and Draq-7.  Cardiac myocyte (CM) nuclei (PCM1+, PLN+, Draq-7+) were purified using a SH800S sorter (Sony Biotechnology).   At least 50,000 CM nuclei were sorted per sample and processed immediately for in situ Hi-C using the Arima Genome-wide Hi-C Kit following instrucitons for mammalian cells. DNA was sheared to an average fragment size of 400 bp with a Bioruptor (Diagenode, 30 cycles, 30 s, on, 90 s off, ‘low’ mode).  Sequencing libraries were then constructed using the Swift Bioscience Accel-NGS 2S Plus DNA Library Kit and amplified with KAPA-SYBR Fast DNA amplification reagents (Kapabiosystems) supplemented with EvaGreen (1x, Biotium) for real-time monitoring.
description
adult
developmental stage
low fat diet
diet
control
disease
wild type genotype
genotype
not collected
geographic location (country and/or sea)
not applicable
isolate
Mus musculus
organism
Mus musculus
organism
heart
organism part
3
replicate
Mus musculus
scientific_name
male
sex
C57BL6N
strain
WT_LFD_#436
title