Illumina HiSeq 2000 paired end sequencing; Functionally conserved non-coding regulators of cardiomyocyte proliferation and regeneration in mouse and human
Functionally conserved non-coding regulators of cardiomyocyte proliferation and regeneration in mouse and human
P0.5 neonatal C57BL6J mice were used for the LAD-ligation model described previously (Haubner et al., 2012). Following hypothermia anaesthesia animals were taped to a cooling bag in the right lateral position. Skin was cut few millimetres below the left foreleg. Next, the Mm. pectorals major and minor were dissected and the thorax opened in the 4th intercostal space. Ligation of the LAD artery was performed with 10.0 ethilon suture (Ethicon). The musculoskeletal thorax and the skin were closed with 8.0 vicryl suture (Ethicon). Finally, mice were placed onto a 38oC heated pad and, after confirmation of spontaneous breathing; a dose of subcutaneous buprenorphine (0.05 mg/kg) was applied. The overall procedure took approximately 10 minutes per animal. Animal survival rates were above 90 % for sham-operated and LAD-ligated mice. Left ventricular tissue distal to the ligature corresponding to the apical third of the left ventricle was harvested after three, five, seven and 10 days following the surgery in 3 animals at each time point. For all the timepoints we also performed sham operation, in which the thorax was opened and the needle was passed under the LAD without its ligature. We also harvested physiological, uninjured left ventricular tissue from the same anatomical location as the samples described above, from one, three, five, seven, and 10 day old mice. All animal experiments were approved by the Austrian Ethical Board.  RNA was extracted from 3 mg of LV tissue from three P1, P3, P5, P7, P10, sham-operated and LAD-ligated LVs using Trizol (Invitrogen) according to manufacturer's instructions with an additional purification step by on-column DNase treatment using the RNase-free DNase Kit (Qiagen) to ensure elimination of any genomic DNA. The integrity and quantity of RNA was determined using Qubit (Invitrogen) and Agilent 2100 Bioanalyzer (Agilent Technologies).  1 µg of total RNA was used to generate RNA-Seq libraries using TruSeq Stranded total RNA sample preparation kit (Illumina) according to the manufacturer's instructions. Briefly, cytoplasmic and mitochondrial rRNA was removed using biotinylated, target specific oligos combined with Ribo-Zero rRNA removal beads. Following purification, RNA was fragmented using divalent cations under elevated temperature and cleaved fragments were copied onto first strand cDNA using reverse transcriptase and random primers. Next, the second strand was synthesised using DNA Polymersae I and RNase H. cDNA 3' ends were adenylated and indexed adapters ligated followed by 10 cycles of library amplification. Finally, the libraries were purified using AMPue XP Beads (Beckman Coulter) and quality was evaluated using Agilent 2100 Bioanalyzer. Three libraries were run on a single lane of the HiSeq 2000 platform (Illumina) to generate 100 bp paired-end reads.