title: GSM1701614: ZT16-24h_Control-R2; Glycine max; RNA-Seq
library_construction_protocol: Total RNA was extracted from leaves using the RNA Plant Reagent® (Ambion) and treated with DNase (Invitrogen). Following analysis of RNA quality and integrity in a Bioanalyzer (Agilent) (RIN ≥ 8.0), equimolar quantities of purified total RNA samples from each of two biological replicates were pooled into one template for library synthesis. For each time period and treatment were synthesized three independent libraries using the Ovation RNA-Seq® (NuGEN Technologies) method, which is suitable to use with the Illumina platform. Approximately 150 ng of total RNA was mixed with the selected primers to synthesize the first strand of the cDNA using a reverse transcription polymerase. The second strand was synthesized using a single primer for reverse transcription and incorporation of analog nucleotides, followed by RNA strand degradation. Double-stranded DNA was fragmented by sonication to a median size of 200 bp and purified with the Agencourt RNAClean XP system (Beckman Coulter) using magnetic beads. Fragment ends were repaired to produce blunt ends and ligated to a pair of double-stranded fragments containing nucleotide analog-tagged adaptors. Strand selection eliminated sequences with the nucleotide analog (insert or insert + adaptor), thereby creating a sequence library with a single insert orientation. Sequences containing both adaptors were amplified using forward and reverse primers for 15 cycles, resulting in a specific-strand, rRNA-depleted cDNA library. Following qualitative and quantitative analysis using a Bioanalyzer, libraries were used to produce clusters for a 2 x 50 bp paired end-sequencing run.
identifiers.namespace: GEO
identifiers.id: GSM1701614