Study : Next Generation Sequencing to Study LUX-affected genes.


Next Generation Sequencing to Study LUX-affected genes.
Purpose: The goals of this study are to compare RNA-seq profiles of Col-0, acd6-1, Iux-1, and acd6-1lux-1 to identify genes affected by LUX. Methods: Total RNA was extracted from 25-d old Col-0, lux-1, acd6-1, or acd6-1lux-1 plants collected at ZT1 or ZT13. Triplicate biological samples were used for most genotypes at each time point, except acd6-1 and acd6-1lux-1 at ZT13, which had duplicate samples. 0.5 ug RNA per replicate was used to generate cDNA libraries using Illumina TruSeq RNA sample preparation kit (catalog no. RS-122-2001). The samples were multiplexed and sequenced using the Illumina HiSeq sequencing platform in Genomics Resources Core Facility at Weill Cornell Medical College. Sequencing was conducted with a standard run of 51 cycles and single reads. At least 150 million reads per lane were obtained for sequencing. qRT–PCR validation was performed using TaqMan and SYBR Green assays for some selected genes. Results: We found that over 1500 genes are affected by lux-1 based on the RNAseq analysis. GO analysis revealed that LUX-affected genes are enchriched in response to abiotic and biotic stimuli. In particular, genes involved in basal defense, salicyclic acid signaling, and jasmonic acid signaling are affected by lux-1. We also found LUX regulation of the clock genes, including core clock components and those acting in the output pathways. Conclusions: The RNAseq analysis support a role of LUX in regulation of the circadian clock and plant defense. Overall design: 25-d old Col-0, lux-1, acd6-1, or acd6-1lux-1 plants were collected at ZT1 or ZT13 for RNA extraction followed by cDNA library preparation and high throughput.


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