alias: GSM2734729
title: GSM2734729: Mid-maturation stage whole seed; Glycine max; RNA-Seq
library_construction_protocol: Collected tissues were quickly frozen in liquid nitrogen and ground to a fine powder using a mortar and pestle. Total RNA was isolated from the powder using the Concert Plant RNA Reagent (Invitrogen, Carlsbad, CA) according to the manufacturerâs instructions. Isolated total RNA was treated with RNase-free DNase I (Ambion, Austin, TX) and was subjected to two rounds of poly-A+ RNA selection using oligo-d(T)25 magnetic beads (Dynabeads, Invitrogen, Carlsbad, CA). Sequencing libraries were prepared using 100 nanograms of twice-selected polyA+ RNA following the Illumina mRNA-Seq Protocol (Part #1004898 Rev. D). Briefly, polyA+ RNA was fragmented and used as template for random-primed cDNA synthesis. Double-stranded cDNAs were treated with T4 and Klenow DNA Polymerases and T4 PNK to generate blunt-end cDNAs. An 'A' base was added to the 3â ends of the phosphorylated blunt-end cDNAs, followed by the ligation of Illumina adapters. Adapter-ligated cDNAs were size selected on an agarose gel for the desired size of ~ 200 bp. Purified cDNAs were amplified by PCR for 15 cycles. Quantification of cDNA libraries was carried out using a Nanodrop ND-3300 Fluoro-spectrophotometer (Thermo Scientific, Waltham, MA). Amplified cDNA size was determined by agarose gel electrophoresis. Single-end 76-bp reads were generated for each library by the UCLA Genome Sequencing Center (http://gsc.ucla.edu/) using an Illumina Genome Analyzer IIx and HiSeq 2000.
identifiers.namespace: GEO
identifiers.id: GSM2734729