LIS08 diel_T19
Dinoflagellate spliced leader-based 454-pyrosequencing technique was employed to generate time-serial ESTs throughout a diel cycle during an Alexandrium fundyense bloom in Long Island Sound. RNA samples from four time points T4, T13, T17 and T19 (hours since sunrise) of the harvested cells were chosen for sequencing.      For each time point, 0.3-0.9 L surface seawater for RNA analysis was filtered onto a 5-micrometer pore size (47mm diameter) polycarbonate membrane using a hand vacuum pump under low vacuum pressure. The membrane was immersed in 1 mL TRIzol reagent (Invitrogen) and kept on ice until transport to the laboratory where the samples were kept at -80°C until RNA extraction. Total RNA was isolated following Zhang et al. 2007 using RNeasy Plus Mini Kit (QIAGEN). The 1st-strand cDNA from each sample was synthesized with an oligo primer named 454BT7N9 (5’- CTATGCGCCTTGCCAGCCCGCTCAGTAATACGACTCACTATAGGGAGNNNNNNNNN- 3’, where the underlined is 454 sequencing primer A and N is any of the 4 nucleotides). The synthesized 1st-strand cDNA was used as the template for PCR amplification of the transcriptome using 454BT7 (5’- CTATGCGCCTTGCCAGCCCGCTCAGTAATACGACTCACTATAGGGAG-3’) as the reverse primer paired with a modified DinoSL primer (454ADinoSL, 5’- CGTATCGCCTCCCTCGCGCCATCAGNCCGTAGCCATTTTGGCTCAAG- 3’ where the underlined is 454 sequencing primer A) as the forward primer. The PCR amplification was carried out for each library under a touch-down PCR program using ExTaq DNA polymerase (Takara Bio): 94 ºC for 1 min; 95 ºC for 15 sec, 60 ºC for 30 sec, 72 ºC for 2 min for 10 cycles; 95 ºC for 15 sec, 52 ºC for 30 sec, 72 ºC for 2 min for 10 cycles. The cDNA amplicons with size range from 500-700 bp were selected by agarose gel cutting and purified. Size-selected cDNA amplicon from each library was sequenced separately by 454 GS FLX Titanium system at the Center for Applied Genetics and Technology, University of Connecticut.