The sampels were ground under liquid nitrogen. From the tissue powder total RNA was isolated using s CTAB based protocol. The total RNA preparations were ananlysed for theit integrity by capillary electrophoresis. Form total RNA polyA+ RNA was prepared which was used for cDNA synthesis. First-strand cDNA was synthesised using an oligo(dT)-linker primer and M-MLV H- reverse transcriptase. The reaction conditions were chosen such that the lenght og first-stand cDNA was limited to ~ 500-1000nt. Finally, the cDNA was amplified (17 cycles) using a high fidility DNA polymerase. For Titanium sequencing the cDNAs in size range of 500-650 bp were eluted from a preparative agarose gel. Aliquots of the size fractionated cDNAs were analzyed by capillary electrophoresis. 5'-454 Adapter A1 and 3'-454 Adapter B1 were ligated and samples tagged bud (TCTACT bud/ TGTATC wt) .
GEO Accession
GSM1465022