GSM2070588: Emerging_Needles; Pinus pinaster; RNA-Seq
The Laser Capture Microdissection (LCM) procedure was carried out as previously described in Cañas et al. (Tree Physiol. 34:1278-1288). The microdissection samples were placed into the caps of 0.5 mL tubes containing 10 L lysis buffer from the RNAqueous-Micro RNA Isolation Kit (Ambion, USA). Before RNA extraction all the samples were placed at -80ºC. All the RNA extractions from the microdissection of the samples were made using the standard-volume protocol (non-LCM) for the RNAqueous-Micro RNA Isolation Kit (Ambion, USA). RNA quality and first quantification was assessed using the RNA Pico Assay for the 2100 Bioanalyzer (Agilent, USA). The RNA samples with a RNA integrity number (RIN) higher than 7 were used for the subsequent mRNA amplification and 454 pyrosequencing. As the amounts of RNA isolated form LCM samples were not enough for 454 pyrosequencing, a previous cDNA synthesis and amplification was made using the Conifer RNA Amplification (CRA+) protocol described in  Cañas et al. (Tree Physiol. 34:1278-1288). Transcriptome sequencing was performed at the Universidad de Málaga ultrasequencing facility using the GS-FLX+ platform with a GS FLX Titanium kit, Roche Applied Sciences (Indianapolis, IN, USA). Each sample was run in one half of a 454 PicoTiterPlate following the manufacturer’s sequencing protocol. The quantity of the cDNA libraries was determined using the Quant-iT™ PicoGreen® dsDNA Kit (Invitrogen, Paisley, UK). The quality of the cDNA libraries was determined using the Agilent High Sensitivity DNA Kit in the 2100 Bioanalyzer (Agilent, CA, USA). The runs were analysed using the Roche GS-FLX+ software.