PMA +/- NFkB inhibition in adult rat cardiomyocytes
Description
Rat cardiomyocytes (adult female Wistar rats) were isolated and kept in culture for a few hours; NFkB inhibitors were added 1 h prior to the addition of PMA (50 nM) and then kept in culture for another 4 h (total culture time: 5 h). Control cells (CTRL) were treated with vehicles only for 5 h. NFkB inhibitors and concentrations were TPCA1 (10 µM) + NIKSMI1 (0.5 µM). The group is named NTP (NIKSMI1+TPCA1+PMA).
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Steps to reproduce
After cell isolation and culture, the viability of cardiomyocytes was verified by light microscopy. If the fraction of rod-shaped myocytes with visible cross striations was at least 70%, cells were used for RNA sequencing, otherwise they were excluded. Freshly isolated cardiomyocytes were subjected to their respective treatments and cultured; inhibitors were incubated for 1 h prior to PMA addition and then 4 h together with PMA for a total of 5 h, all vectors were accordingly added to the respective controls. Cells were collected in their respective media and centrifuged at 2000rpm for 2 min, after which the supernatant was discarded and the pellet was frozen and stored at -80 °C until use. Total RNA was extracted using a commercial kit (Nucleospin, Machery Nagel, Germany) according to the manufacturer’s instruction. Samples from one isolation were handled in parallel to minimize batch-effects between groups. After extraction purity was controlled by measuring 260/280 and 260/230 absorbance ratios by spectrophotometry (DS-11+ Spectophotometer, DeNovix, Wilmington USA). All samples contained more than 100ng of RNA. RNA was then frozen at -80°C until handing them over to the sequencing facility. A second quality check using a commercial device (BioAnalyzer, Agilent, Santa Clara, USA) was used to exclude samples with degraded RNA. The RNA was processed into a library with the Illumina Stranded mRNA Kit (Illumina, San Diego, USA). The library was sequenced as 150+150bp paired-end reads on an Illumina Novaseq 6000 platform. Each sample yielded 32-57 million raw reads. After removal of optical and proximal duplicates, adapter and quality trimming, the resulting reads were aligned against the mRatBN7.2 reference genome with the STAR aligner software (version 2.7.10 a)47. Transcript quantification was performed with Salmon 1.9 software. Statistical analysis of normalized pseudocounts was carried out with the DEseq2 package (v1.44) in the R statistics environment (v4.4.2) library.
Institutions
- Friedrich-Alexander-Universitat Erlangen-Nurnberg