454 GS FLX sequencing; Emiliania huxleyi
Axenic cultures of pure diploid, highly calcified cells (strain is RCC1216) were grown in natural seawater supplemented with modified K/5 nutrients under 80 mol photons/m2/s light on a 14:10 light:dark cycle. Cells were harvested at 8 time points over a 24 hour period beginning when cell abundance was approximately 200000 cells/ml (mid-exponential growth).  Nutrients were not limiting at this point. Equal amounts of total RNA was pooled from each time point. RNA from each time point and from the pooled sample was assessed to be of high quality and not degraded based on analysis on a Agilent Bioanalyzer showing clear distinct 28s and 18s ribosomal RNA bands and spectrophotometric determination of high A260/A280 ratios. RT-PCR using several transcribed genes confirmed the absence of detectable genomic DNA contamination.
cDNA preparation was performed by Vertis, Inc. From the total RNA poly(A)+  RNA was prepared. First-strand cDNA synthesis was primed with a N6 randomized primer. Then 454 adapters A and B were ligated to the 5' and 3' ends of the cDNA. The cDNA was finally amplified with PCR using a proof reading enzyme (22 cycles). For 454 sequencing the cDNA in the size range of 450 – 650 bp was eluted from a preparative agarose gel using the Macherey and Nagel NucleoSpin Extract II kit. The cDNA has a size of about 450 – 650 bp and exhibits the following structure:5'-454 Adapter A GCCTCCCTCGCGCCATCAG- 5’-Adapter cDNA (350-550 bp) 3’-Adapter CTACTAGACCTTGGCTGTCACTCANNNNNNNNNNNNNNNNNNNNNNNNNNNNNNTCGCAGTGAGTGACAGGCTAGTAG 3'-454 Adapter B - CTGAGCGGGCTGGCAAGGC