Sample of Coffea arabica Rubi non-irrigated
Plagiotropic buds were incubated for 5 min. in the washing buffer (66% chloroform, 33% methanol, 1% HCl) and further incubated twice (30 min.) under vacuum in the fixation buffer (25% acetic acid, 75% ethanol RNAse-free) cooled at 4 °C, changing the buffer between each incubation. Samples were stored in 75% ethanol RNAse-free. Apices were separated under binocular microscope dissection from plagiotropic buds from three different plants for each condition and then ground to powder in liquid nitrogen using a mortar and pestle. Total RNAs were extracted using the Nucleospin RNA Plant kit (Macherey-Nagel), including a DNase-I treatment. Quality and quantity of RNAs were checked with a Bioanalyzer (2100, RNA Nano 6000 Agilent). The 1st strand cDNA synthesis was performed using 1 μg total RNA and following the SMARTer cDNA Double-stranded cDNA was then produced using 22 to 25 independent 2nd strand synthesis reactions and quantify with by Bioanalyzer (Agilent).   cDNA library was constructed, corresponding to the Rubi cultivar subjected to non-irrigation. For this sample, cDNA (about 5 μg) was nebulized to a mean fragment size of 650 bp and ligated to an adaptor, according to standard procedures. cDNA sequences were then sequenced performing 2 runs (1 library per cDNA sample x 2) using GS-FLX Titanium (Beckman Coulter Genomics SA, Grenoble, France) that generated around one million of reads corresponding to more than 255 Mb.