alias: E-MTAB-2997:S-Set
title: Illumina HiSeq 2000 sequencing; Comprehensive identification of miRNAs involved in eucalypt xylogenesis
design: Comprehensive identification of miRNAs involved in eucalypt xylogenesis
library_construction_protocol: Differentiating xylem (DX) tissues were harvested after detaching the bark, by quickly scratching the exposed soft gelatinous cell layers (2-3 mm thick) using a clean scalpel blade. The tissues were collected into sterile flasks previously cooled with liquid nitrogen, transported on dry ice avoiding defreeze and stored at -80C until use. The sampling campaign of E. globulus seasonal wood DX-tissues was done in collaboration with RAIZ Institute (Portugal). Four sampling time points were performed during the year of 2008: February 26 th (Feb), May 23rd (May), September 5th (Sep) and December 3rd (Dec). A population of thirty six trees, four years old, from three non-related distinct genotypes (HD161, CN5 and G-70) was selected at Herdade do Zambujal, Pegoes (Portugal). Single samples of DX-tissues were harvested from three ramets of each genotype in each time point. All ramets were previously certified in terms of clonal identity using a battery of seventeen microsatellites. Total RNA was isolated using approximately 100mg of biological material per sample. A modified protocol after [][V1]  was used by omitting the LiCl precipitation and by precipitating the RNA for one hour at -20C following the addition of 0.1X (v/v) NaOAc 4M (pH 5.2) and 2.5X (v/v) of absolute ethanol. RNA integrity was visualized on 2% agarose 0,5X TBE gels. For the construction of small RNA (smRNA) libraries equimolar pools of total RNA were produced from high quality individual total RNA samples from the SW2008 and RW2010 campaigns following a strategy adjusted to the correspondent model of study. Fifteen micrograms of each total RNA pool was sent to FASTERIS SA (Geneve, CH; www.fasteris.com) for smRNA libraries preparation and sequencing. For library preparation, RNA was processed in vitro to generate a library of short inserts (DNA 22 Colonies Template Library). The quality of these libraries, was checked by cloning an aliquot of the library into a vector, and sequences the inserts by Sanger technology.
identifiers.namespace: IBET - INSTITUTO DE BIOLOGIA EXPERIMENTAL E TECNOLOGICA
identifiers.id: E-MTAB-2997:S-Set
identifiers.namespace: iBET - Instituto de Biologia Experimental e Tecnologica
identifiers.id: E-MTAB-2997:S-Set