GSM2717014: MgCl2_day3_10h_1; Arabidopsis thaliana; RNA-Seq
Leaf harvests at day 3 were done at 10 h and 15 h after dawn and at day 4 at 9 h after dawn. Frozen leaf tissue (40 - 100 mg) from three independent replicates each of bacteria-primed and mock treated plants was ground (2 x 45 s, maximum frequency) by a Retsch mill (MM 400, Retsch, Haan, Germany). Each harvest was a single young systemic leaf from a single rosette. The harvested systemic leaves were all developmentally younger than the previously locally infected leaves. RNA was extracted with the RNeasy Plant Mini Kit according to the manual (QIAGEN GmbH, Hilden, Germany). RNA was quantified with a Qubit® RNA HS Assay Kit (ThermoFisher Scientific Life Technologies GmbH, Darmstadt, Germany) and RNA quality was further assessed by agarose gel electrophoresis. Library preparation and sequencing was done at the Max-Planck-Genome Centre (Cologne, Germany) according to the manufacturer's protocols. Briefly 500 ng of mRNA was poly-A enriched by applying the NEBNext Poly(A) mRNA Magnetic Isolation Module and the NEBNext Ultra directional RNA library prep kit for Illumina was used to construct the library before amplification in 12 PCR cycles. Sequencing was performed on a Illumina HiSeq 3000 sequencer.