Microarray Analysis of Cardiomyocyte RNA

RNA isolation of cardiomyocytes was carried out using the NucleoSpin® RNA isolation kit (Macherey–Nagel, Düren, Germany) according to the manufacturer instructions. RNA integrity was evaluated using the Agilent Bioanalyzer 2100 with the RNA Pico chip kit (Agilent Technologies). For microarray analysis, 200 ng of isolated RNA samples were applied, and hybridization was performed as described previously [26 (link)]. Hybridization was carried out on Affymetrix Clariom™ D Arrays according to the instructions of the manufacturer (Thermo Fisher Scientific). Data analysis was conducted with the provided Transcriptome Analysis Console Software from Thermo Fischer Scientific (Version 4.0.1). The raw microarray data is accessible at the EMBL-EBI ArrayExpress website under the accession number E-MTAB-13152 (https://www.ebi.ac.uk/biostudies/arrayexpress/studies?query=E-MTAB-13152) [27 ]. The analysis included quality control, data normalization, and statistical testing for differential expression (Limma). Transcripts are considered as significantly differentially expressed (DE) with a fold change (FC) higher than 2 or lower than − 2, false discovery rate (FDR) < 0.5, and p-value < 0.05. Gene expression networks were visualized using the Cytoscape application ClueGo [28 (link)]. Heatmaps were generated using heatmapper [29 (link)].