De novo assembly of perennial ryegrass transcriptome using an RNA-seq strategy
Total RNA was extracted from each sample using the RNeasyTM Plant Mini Kit following the manufactures instructions (Qiagen, Valencia, CA) and the RNA integrity was measured with an RNA 6000 Nano LabchipTM on the Agilent 2100 BioanalyzerTM (Agilent Technologies, Santa Clara, CA). Using the mRNA-Seq Sample Prep Kit (Illumina, protocol version 1004898 Rev. D, September 2009), the mRNA-Seq library was generated using 10 µg total RNA from each of the twelve samples; it was purified using poly-T oligonucleotide-attached magnetic beads and fragmented using divalent cations under elevated temperature. Using reverse transcriptase and random hexamer primers, first strand cDNA copies of the mRNA fragments were generated. The second strand cDNA was then synthesized using DNA Polymerase I and RNaseH. Using the Multiplexing Sample Preparation Oliogonucleotide KitTM, the Illumina adaptors were added by ligation and a fragment size of approximately 200bp was isolated via gel purification. The libraries generated were enriched by 18 PCR cycles. Using the InvitrogenTM Qubit® Fluorometer (Life Technologies, Carlsbad, CA), the concentrations of the libraries were determined, and purity and size of the libraries were measured using the DNA 1000 kitTM on the Agilent 2100 BioanalyzerTM (Agilent Technologies, Santa Clara, CA). An equimolar amount of each library was pooled and diluted with EB buffer (Qiagen, Valencia, CA) to 10 nM for preparation for paired-end Illumina GAIIx sequencing.