GSM3389405: salt-24h; Festuca arundinacea; RNA-Seq
Total RNA from the leaves of each sample was isolated using Trizol reagent (Invitrogen, Carlsbad, CA) and purified using the RNeasy Plant Mini Kit (Qiagen, Valencia, CA) following the manufacturer's protocol. The RNA was analyzed using the Agilent 2100 Bioanalyzer (Agilent RNA 6000 Nano Kit). Total RNA concentration with a fragment size of 28S/18S and purity was measured using a UV spectrophotometer NanoDropTM and processed by enriching the mRNA of polyA tails with magnetic beads containing Oligo(dT) followed by the fragmentation of the obtained RNA using an interrupting buffer. Random primers were used for reverse transcription, and cDNA duplex was synthesized to form double-stranded DNA. Subsequently, the synthetic double-stranded DNA was filled at the 5' and 3' ends followed by ligation. The ligation product was amplified by PCR using specific primers. Briefly, the material was heat-denatured into single-stranded chains, and a single-stranded circular DNA library was cyclized with a bridging primer to obtain a single-stranded circular DNA library followed by sequencing on a computer. The raw data sequenced contained low-quality, linker contaminants, and reads with too high and unknown base content. Therefore, to ensure the reliability of results internally developed software was used for filtering out the reads. The filtered data was designated 'clean reads' which were candidates of de novo assembly. 'Trinity' program [31] was used to remove PCR duplicates in order to improve the assembly efficiency of clean reads. The assembled transcripts were then clustered and unigenes were obtained. The unigenes were divided into two parts; the first were clusters, which was the result of further redundancy. (Beginning with CL, denotes cluster, followed by the number of the gene family). The remaining were singletons (beginning with Unigene).
GEO Accession
GSM3389405