454 sequencing of Sus scrofa heart transcript fragment library
PolyA+ RNA was purified from ~200 micro g Sus scrofa heart (HEA) Total-RNA using Oligotex mRNA Mini Kit (Qiagen). The poly A+ RNA isolate was used as template in synthesis of high quality double-stranded cDNA with oligo(dT)12-18-priming of the first strand cDNA (SuperScriptTM Doubled-Stranded cDNA Synthesis Kit, Invitrogen). Final purification of the synthesized cDNA was performed on MinElute PCR Purification column (Qiagen), and an average cDNA fragment size of 300-400 bp was obtained by applying 45 psi (= 3 bar) of nitrogen for 50-60 seconds on 2.7-4.7 micro g starting material. Subsequently, the distribution of fragments was profiled on a BioAnalyzer DNA 1000 LabChip (Agilent Technologies). As a part of the library preparation, the ends of the sheared cDNA fragments were polished by treatment with T4 DNA Polymerase and T4 Polynecleotide Kinase prior to ligation of sequencing adaptors (Margulies et al. 2005). The fragments were immobilized on streptavidin beads, following nick repair with Bst DNA Polymerase, and the sstDNA libraries were eluted by alkali denaturation. Finally, the qualities and quantities of the libraries were assayed on Bioanalyzer RNA 6000 Pico LabChip (Agilent Technologies).