RNA-Seq of mouse: Heart HEP_TAC4
Transcriptomic analysis was performed at the Single Cell Genomics Facility, Cincinnati Childrens Hospital Medical Center. Briefly, the initial amplification step for all samples was done with the Ovation RNA-Seq System v2 kit (Tecan Genomics). The assay was used to amplify RNA samples to create double stranded cDNA. A total of 10 ng RNA was used per sample. The assay created between 3.2 -4.3 micrograms of double stranded cDNA for each sample. The concentrations were measured using the Qubit dsDNA BR assay. The cDNA size of each sample was determined by using an Agilent HS DNA Chip. Libraries were then created for all samples using 1 ng of cDNA. Specifically, the Illumina protocol, the Nextera XT DNA Sample Preparation Kit, was used to create DNA library templates from the double stranded cDNA. The concentrations were measured using the Qubit dsDNA HS assay. The size of the libraries for each sample was measured using the Agilent HS DNA chip. The samples were placed in a pool. The concentration of the pool was optimized to acquire at least 30 million reads per sample by using Paired-End 100 bp Reads, so for sequencing a NovaSeq SP (200 cycles) v1.5 flow cell was used. Libraries were sequenced on the Illumina NovaSeq 6000 system following the manufacturer's protocol.