title: Illumina HiSeq 2500 sequencing; RNA-seq of atspf30-1 vs Col-0 seedlings grown in LL conditions.
design: RNA-seq of atspf30-1 vs Col-0 seedlings grown in LL conditions.
library_construction_protocol: Samples containing 15-30 seedlings were flash frozen in liquid nitrogen. Col-0 and atspf30-1 seeds were sown on Murashige and Skoog medium (GIBCO BRL) and cold stratified (4ºC, darkness) for 3 days. MS plates were then transferred to 22 °C and 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).
attributes.tag: Experimental Factor: genotype
attributes.value: wild type genotype