title: Changes in gene expression of Haematococcus pluvialis following high light intensity and nitrogen starvation
design: Total RNA was isolated according to the instruction of TRIzol kit (Invitrogen) and then purified to exclude the tRNA and rRNA and to enrich mRNA, according to the instruction of mRNA purification kit (Promega). mRNAs were reverse-transcribed by Powerscript™ II (Takara) with PCR primers SMART IV™ Oligonucleotide (5'-AAGCAGTGGTATCAACGCAGAGTGGCCATTACGGCCGGG-3') and CDS III/3' PCR Primer (5'-ATTCTAGAGGCCGAGGCGGCCGACATG-d(T)30N-1N-3'). Long Distance PCR for double strand cDNA amplification was performed with LA Taq enzyme (Takara) for 25 cycles (95°C for 30 s, 68°C for 8 min) according to the SMART™ cDNA Library Construction Kit User Manual. Finally, double strand cDNAs were purified using DNA purification kit (Qigen) to generate high quality cDNAs.   Approximately 10 μg of sheared cDNA were then prepared for Illumina sequencing according to the manufacturer's protocols. Libraries were prepared from a 300–500 bp size-selected fraction following adapter ligation and agarose gel separation. The libraries were sequenced using a paired end read protocol with 100 bp of data collected per run on the Illumina Genome Analyzer.