title: RNA-seq analysis of cardiac fibrosis in mouse hearts 28 days post-myocardial infarction with (MIEru) or without (CONMI) dietary erucic acid supplementation
design: RNA-seq analysis of cardiac fibrosis in mouse hearts 28 days post-myocardial infarction with (MIEru) or without (CONMI) dietary erucic acid supplementation
library_construction_protocol: Total RNA was isolated from approximately 20-30 mg of snap-frozen left ventricular tissue. The tissue was homogenized in 1 mL of TRIzol Reagent (Thermo Fisher Scientific) using a mechanical homogenizer. RNA extraction followed the manufacturer's standard protocol. Briefly, after phase separation with chloroform, RNA was precipitated from the aqueous phase with isopropanol, washed with 75% ethanol, and dissolved in RNase-free water. RNA concentration and purity were assessed using a NanoDrop spectrophotometer, and RNA integrity was verified by agarose gel electrophoresis or Bioanalyzer (RIN > 7.0). RNA-seq libraries were constructed using the NEBNext Ultra II Directional RNA Library Prep Kit for Illumina (New England Biolabs). Briefly, 1 Âµg of total RNA was used for poly(A) mRNA selection using oligo-dT beads. The enriched mRNA was fragmented, followed by first and second strand cDNA synthesis. The double-stranded cDNA was end-repaired, adenylated at the 3' ends, and ligated to Illumina-compatible adapters. Ligated fragments were then amplified by PCR (12-15 cycles) with index primers to generate final libraries. Library quality and size distribution were checked using an Agilent Bioanalyzer High Sensitivity DNA chip. The final libraries were quantified by qPCR, pooled in equimolar amounts, and sequenced on an Illumina NovaSeq 6000 platform (Novogene Co., Ltd. or specify your sequencing facility). Sequencing was performed in a 150-base pair paired-end (PE150) configuration to a target depth of approximately 40 million reads per sample, following the manufacturer's standard sequencing protocol.