alias: E-MTAB-2997:S2WO
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. Reaction wood (RW) formation was induced by bending vertical E. globulus trees to an angle of roughly 45degrees, during different five bending times, at Altri Florestal SAs Experimental Research Station of Quinta do Furadouro-obidos (Portugal). Samples were collected in three certified trees (ramets) of three certified genotypes (GB3, GM2-58 and MB43 genotypes) provided by Altri Florestal SA (Portugal). Five years old trees were bent in June 9th 2010 (4 weeks of bending), June 16th 2010 (3 weeks of bending), June 23rd 2010 (2 weeks of bending), June 30th 2010 (1 week of bending), and July 6th 2010 (1 day of bending), and harvested along with the non-bent trees in July 7th 2010. DX tissues were collected from the upper (tension wood, TW) and the lower (opposite wood, OW) side of the bent trunk segment. Control DX samples (Ctrl) were harvested at the diameter at breast height (around 1.3 m high) from non-bent trees. 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:S2WO
identifiers.namespace: iBET - Instituto de Biologia Experimental e Tecnologica
identifiers.id: E-MTAB-2997:S2WO