Illumina HiSeq 2500 paired end sequencing; RNA-seq of circadian timeseries sampling (LL2-3) of 13-14 day old Arabidopsis thaliana Col-0 (24 h to 68 h, sampled every 4 h)
RNA-seq of circadian timeseries sampling (LL2-3) of 13-14 day old Arabidopsis thaliana Col-0 (24 h to 68 h, sampled every 4 h)
Samples containing 15-30 seedlings were flash frozen in liquid nitrogen. Samples were collected at 4 h intervals, starting at 24 h after transfer to LL until 68 h post-transfer to LL. Col-0 seeds were sown on Murashige and Skoog medium (GIBCO BRL) and cold stratified (4ºC, darkness) for 3 days. The MS plates were transferred to 22 °C under LD12:12 (12 h light/12 h dark cycles; 50 μmol·m−2·s−1 of white light) conditions for 11 days and then transferred to continuous light (LL; 50 μmol·m−2·s−1 of white light). Total RNA was extracted using the Trizol method according to the manufacturer's protocol (ThermoFisher) and quantified using a NanoDrop spectrophotometer (ThermoFisher). Total RNA integrity was further checked by agarose gel electrophoresis. Libraries were prepared following the TruSeq RNA v2 Sample Preparation Guide (Illumina). Briefly, 3 μg of total RNA was polyA-purified and fragmented, and first-strand cDNA synthesized by reverse transcriptase (SuperScript II; Invitrogen) and random hexamers. This was followed by RNA degradation and second-strand cDNA synthesis. End repair process and addition of a single A nucleotide to the 3′ ends allowed ligation of multiple indexing adapters. Then, an enrichment step of 12 cycles of PCR was performed. Library validation included size and purity assessment with the Agilent 2100 Bioanalyzer and the Agilent DNA 1000 kit (Agilent Technologies).
Experimental Factor: time
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