title: The transcriptional mechanism behind Mimosa pudica leaf-folding in response to physical disturbance
design: The transcriptional mechanism behind Mimosa pudica leaf-folding in response to physical disturbance
library_construction_protocol: Leaves of non-stimulated, stimulated and over-stimulated plants were cut at the base of the hypocotyls and immediately snap frozen in liquid nitrogen. Plants at identical developmental stages (young, 10 cm tall, 5 leaves brunches) were purchased from a local specialized nursery and grown for a week for acclimatation in a growth chamber using controlled conditions of light, temperature (23 °C) and humidity. Three different stimulations were applied to plants, and samples were collected immediately after the treatments described below.  1. Non stimulated plants: untreated plants, with leaves wide open. The leaves were cut at the base of the hypocotyls and immediately snap frozen in liquid nitrogen (these samples were named C) 2. Stimulated plants: plants were subjected to a single a sudden drop from the height of 10 cm. Closed leaves were cut at the base of the hypocotyls and immediately snap frozen in liquid nitrogen (these samples were named T) 3. Overstimulated plants: plants were subjected to repetitive drops from the height of 10 cm, waiting for cycles of closing and reopening of leaves until they did not close/respond to the movement anymore (adaptation). Still open leaves were cut at the base of the hypocotyls and immediately snap frozen in liquid nitrogen (these samples were named M). Total RNA was extracted from samples of Mimosa leaves of plants in control conditions (C) (no external stimuli), of plants after one drop (T), and after several drops until habituation (M), collected as previously reported. For each experimental conditions, three biological replicates were carried out. Samples were immediately frozen in liquid nitrogen and stored at −80 °C. RNA extraction was performed using 100 mg of frozen material ground with a pestle and mortar in liquid nitrogen using the Norgen's Plant/Fungi Total RNA Purification Kit RNA. Final elutions were performed using 60 μL of RNase-free water. RNA was treated with DNAase before quantification. RNA quality was determined using the Agilent 2100 Bioanalyzer System (RNA 6000 nano kit - Biorad Inc.) Sequencing libraries were obtained following the procedure of Truseq Stranded mRNA Library Prep (Illumina) and using Truseq RNA Single Indexes (3 libraries for each of the three experimental conditions). The concentration of each library was determined using Qubit™ 4 Fluorometer (dsDNA High Sensitivity Kit - Invitrogen).