miRNA of Suaeda aralocaspica seedlings by HiSeq 2000
Suaeda aralocaspica developing a unique combination of dispersal and germination strategies via producing dimorphic seeds is thought to be a bet-hedging strategy advantageous in harsh and unpredictable environments. With the advent of post-genomics technologies, large-scale gene expression profiling has been implemented at the RNA, protein and metabolite levels to unveil specific features of the complex germination process. Genome-wide expression studies in Arabidopsis have been previously applied to gain insight into temporal and spatial changes during Arabidopsis germination and provide important new information about mechanisms controlling germination. Nevertheless, a detailed knowledge of the temporal changes in gene expression in halophyte seed is far missing, but it is essential to understanding the control of the timing of germination as well as the underlying molecular processes contributed by the bet-hedging germination of desert annuals. Therefore, we have analyzed the Suaeda aralocaspica transcriptome by sampling 3 points along the germination time course. Our high throughput data set will establish meaningful connections linking genetic information to the physiological changes observed during halophyte germination.  For small RNA-seq, total RNA was extracted from each sample. Three μg of each RNA sample was used for small RNA cDNA library preparation with Balancer NGS Library Preparation Kit for small/microRNA (GnomeGen, San Diego, CA, USA) based on manufacturer’s instruction. Briefly, RNAs were ligated to 3’ and 5’ adaptor sequentially, reverse transcribed to cDNA and PCR amplified. Whole library was applied to 10% native PAGE gel electrophoresis and bands corresponding to miRNA insertion were cut and eluted. After ethanol precipitation and washing, the purified small RNA libraries were quantified with Qubit Fluorometer (Invitrogen, Carlsbad, CA, USA) and used for cluster generation and applied to Illumina GAIIx (Illumina, San Diego, CA, USA) 73nt single-end sequencing and Illumina HiSeq 2000 100nt single-end sequencing according to the manufacturer’s instructions.