Pyrosequencing cichlid fish Amphilophus zaliosus ESTs
Poly A RNA was prepared from total RNA. First-strand cDNA synthesis was performed using oligodT adapter primer with reverse transcriptase. Second strand synthesis was done by N6 random adapter primer. cDNAs were amplified. Normalization was done with one cycle of denaturation and reassociation and then ss-cDNA was separated using a hydroxylapatite column. ss-cDNA was amplified. cDNA in size range of 450-550bp was selected. Each cDNA is structured: 5'-454_Adapter_A_GCCTCCCTCGCGCCATCAG-5'_adapter_CTACTAGACCTTGGCTGTCACTCA then 350-450 BP cDNA then the 3'_adapter_TCGCAGTGAGTGACAGGCTAGTAG-3'-454_adapter_B_CTGAGCGGGCTGGCAAGGC Prepared and sequenced by eurofins MWG Ebersberg German November 2008 using GS FLX standard chemistry.