Cannabis sativa (cv. Santhica 27) hypocotyls of 6, 9, 15, and 20 days after sowing were grown in controlled conditions in incubators following a cycle of 16 h light 25°C/8 h dark 20°C. Three biological replicates per time point were analyzed. Each biological replicate consisted of 20 hypocotyls randomly selected among all incubators. The pooling of hypocotyls was needed because of the quantity of material required for transcriptomics. In order to bring to a minimum this pooling bias, a high number of hypocotyls were pooled together. By doing so, the power to detect differentially expressed genes within the four populations increased (Rajkumar et al., 2015 (link)). Sampling was performed on a single experimental batch to minimize technical variability. Samples were immediately frozen in liquid nitrogen and conserved at -80°C until RNA extraction. The sampled hypocotyls were crushed to a fine powder using a mortar, a pestle and liquid nitrogen. Total RNA was extracted using the RNeasy Plant Mini Kit (Qiagen), treated with DNase I on column, quantified and quality-checked using a Qubit 2.0 Fluorometer (Invitrogen) with the Qubit RNA Assay Kit (Molecular Probes), a NanoDrop 1000 Spectrophotometer (Thermo Scientific) and a 2100 Bioanalyzer (Agilent Life Sciences). All the RNAs displayed a RIN above 7.
Free full text: Click here
