title: Illumina HiSeq 4000 paired end sequencing; RNA-Seq of rat myocardial slices cultured with biomimetic electromechanical stimulation
design: RNA-Seq of rat myocardial slices cultured with biomimetic electromechanical stimulation
library_construction_protocol: Myocardial slices were prepared from 3 male Sprague Dawley rats. 0 Hr slices were flash-frozen immediately following preparation in liquid nitrogen. Unloaded slices were cultured on Transwell membranes (no electromechanical stimulation) and electromechanically stimulated slices were cultured at a sarcomere length of 1.8 or 2.2 microns for 24 hours and then flash-frozen.  Following culture, myocardial slices were immediately frozen in liquid nitrogen and stored at -80oC. A TissueLyser LT (Qiagen) was used to disrupt the sample. The frozen slices were transferred to a pre-cooled 2mL round-bottom tube containing a 5mm stainless steel ball and 500μl TRIzol (Thermo Fisher Scientific). The TissueLyser was set to 40Hz and left for 3-5 minutes until the sample was completely lysed. 100μl chloroform (Sigma) was added each tube and then vortexed for 15 seconds. The tube was then left in a rack for 5 minutes at room temperature and intermittently shaken. The tube was then centrifuged at 12879 x g at 4oC for 15 minutes and phase separation occurred. The top aqueous layer (containing the RNA) was then removed. An equivalent volume (to that of the aqueous layer removed) of 70% ethanol was then added and the solution mixed via pipetting. All of the solution was then transferred to an RNeasy spin column (Qiagen) and the manufacturer's instructions were then followed. Following collection, RNA was frozen and kept at -80oC.  Nucleic acid library construction was performed using TruSeq Stranded mRNA.