title: RNA-seq of mus musculus: adult female heart
design: Total RNA was extracted from snap frozen murine hearts using RNeasy Midi Kit (QIAGEN, 75144).  Basically, tissue was pulverized using mortar and pestle, an appropriate amount of RLT buffer was added, and tissue was homogenised by using 20-gauge needle fitted to an RNase-free syringe.  Contaminating proteins and DNAs were removed using proteinase K and RNAse free DNAse, respectively.  RNA was eluted in a fresh collection tube with RNase-free water.  Total RNA was quantified using Quant-IT RiboGreen RNA Assay Kit (ThermoFisher Scientific, InvitrogenTM, R11490), and the integrity of RNA was assessed by a high sensitivity RNA ScreenTape (Agilent Technologies, 5067-5579).  RNA integrity number estimates for all samples were over 8.  Purification of mRNA, generation of double stranded cDNA and library construction were performed using NEBNext UltraTM II Directional RNA Library Prep Kit for Illumina (New England Biolabs, E7760) with minor modifications to manufacturer specifications.  Oligo d(T) beads were used to enrich mRNA with poly (A) tails.  RNA was fragmented followed by first and second strand cDNA synthesis.  Fragments were end repaired and A-tailed followed by adapter ligation.  Finally, there was a USER-enzyme digestion and PCR enrichment step.  Amplified libraries were analyzed for size distribution using the Agilent Tapestation 2200 D1000.  Libraries were quantified using Picogreen and relative volumes were pooled accordingly.  Sequencing was performed as 150bp paired end read on a NovaSeq6000 platform.