GSM1838957: t0_2; Arabidopsis thaliana; RNA-Seq
Total RNA was isolated with the RNeasy Plant Mini Kit (Qiagen) and 8 µg of RNA were DNase treated with DNA-freeTM Kit (Ambion). Library preparation was performed using the Truseq stranded mRNA sample preparation kit (Illumina, ref.RS-122-2101 ) according to the manufacturer instructions. Briefly, polyadenylated RNAs were purified using oligo-d(T) magnetic beads, fragmented, and reverse transcribed using random hexamers, Super Script II (Life Technologies, ref. 18064-014) and Actinomycin D. During the second strand generation step, dUTP substituted dTTP. This prevented the second strand to be used as a matrix during the final PCR amplification. Double-stranded cDNAs were adenylated at their 3' termini before ligation was performed using Illumia's indexed adapters. Ligated cDNAs were amplified following 15 PCR cycles and PCR products were purified using AMPure XP Beads (Beckman Coulter Genomics, ref.A63881). Libraries were validated using a DNA1000 chip (Agilent, ref. 5067-1504) on a Agilent Bioanalyzer and quantified using the KAPA Library quantification kit (Clinisciences, ref. KK4824). Equimolar amounts of 4 libraries were pooled, diluted to 10 nM, denatured, and diluted again to 7 pM. 100 µl of the diluted pool were hybridized on one lane of an Illumina's Flow Cell. Clustering and 50 cycles sequencing were performed according to Illumina's instructions on a Hiseq2000.
GEO Accession
GSM1838957