GSM1972498: lec1-1 mutant Arabidopsis seed 15daf; Arabidopsis thaliana; RNA-Seq
For RNA-Seq total RNA was isolated using the TRIZOL reagent (Invitrogen) according to the manufacturer's instructions and further purificated using the Qiagen RNeasy plant mini kit according to the manufacturer's instructions. Five μg of RNA from each experimental condition was pooled to obtain a single RNA sample. First and second strand cDNA synthesis was performed with 3 μg of the total RNA mixture using Message Amp-II kit (Ambion) following the manufacturers recommendations. Each sample was treated separately 10-12 ng of synthesized cDNA was amplified by in vitro transcription and the resulting 70-90 μg of antisense RNA (aRNA) purified using RNAeasy columns (Qiagen). A second round of cDNA synthesis was performed using the mRNA as template (20 μg). cDNA synthesis was performed as described above except that random primers (mostly hexamers) were used at the first strand synthesis stage. This procedure yielded approximately 10 μg of cDNA that was purified using the DNA Clear Kit for cDNA purification (Ambion). Samples were barcoded for multiplexing using the SOLiD RNA Barcoding Kit