CCMP1719 diel_T4
Dinoflagellate spliced leader-based 454-pyrosequencing technique was employed to generate time-serial ESTs throughout a 24-h diel cycle of Alexandrium fundyense CCMP1719. RNA samples from four time points T4, T13, T17 and T19 (hours since lights on) of the harvested culture cells were chosen for sequencing.    A. fundyense strain CCMP 1719 was obtained from National Center for Marine Algae and Microbiota. Cultures were grown in f/2 (-Si) medium using 0.2µm- filtered seawater (salinity 28PSU) at 15°C under 14:10 light:dark cycle with a photon flux of 110 ± 10 µ Em-2 s-1. The cultures were synchronized at early G1 phase by the dark-induced block/release method. The diel sampling was conducted over a 24-h period on the 3rd day after the re-exposure to the light-dark cycle. Sampling started 1 h after the onset of the light period and continued every 3 h during the light period and every 2 h during the dark period.     For RNA analysis, at each sampling event 500 mL of the culture was filtered onto a 5-µm pore size (47mm diameter) polycarbonate membrane, and the cells were carefully washed off from the membrane with 1mL Trizol and immediately frozen at -80°C until RNA extraction. Cells fixed in Trizol were homogenized on a Fastprep-24 Tissue and Cell Homogenizer (MP Biomedicals) using Lysing Matrix C (~1mm) silica beads at speed of 6 m/s for two 40-second cycles. Total RNA was isolated following Zhang et al. 2007 using RNeasy Plus Mini Kit (QIAGEN). RNA samples from four time points T4, T13, T17 and T19 corresponding to cell cycle G1 phase, light-dark transition, S phase and  G2M phase in cultures were chosen for 454 sequencing. 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) using Quantitect Reverse Transcription Kit (QIAGEN). 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.
University of Connecticut
CCMP1719 diel_T4