Profiling Small RNA in Cardiac Muscle

A total of 6 samples (isolated from the NRCMs) were used for total RNA extraction using the TruSeq Small RNA Sample Prep Kits (Illumina, San Diego, CA, USA) according to the manufacturer’s protocol. The quality of the RNA samples was examined using a NanoDrop 2000 spectrophotometer (Thermo Scientific, Waltham, MA, USA) and standard denaturing agarose gel electrophoresis. Small RNA library preparation was performed using TruSeq Small RNA Sample Prep Kits (Illumina, San Diego, CA, USA). The quality-ensured RNA-seq libraries were then sequenced using Illumina Hiseq2000/2500. The identification of known miRNAs (mapped to the miRbase database) and read counting were processed using ACGT101-miR (LC Sciences, Houston, TX, USA). A modified normalization was used to correct copy numbers among different samples, and a miRNA was considered present when the normalized read count was >0 in all the samples. A heatmap was constructed using the normalized read counts of the known miRNAs in each EV sample using R (R version 4.0.3) with a heatmap via a custom written R script [46 (link)].