RNAseq of dark dissected, 3 day old Arabidopsis (col0) seedlings, including cotyledons, apical hooks and hypocotyls
RNAseq of dark dissected, 3 day old Arabidopsis (col0) seedlings, including cotyledons, apical hooks and hypocotyls
Three day old etiolated seedlings were grown as described above. Dissections were carried out under dark  466 conditions with a green safe-light. Etiolated seedlings were rinsed in a 1:1 solution of RNA-later (invitrogen)  467 and RNAse free water, before separating cotyledons, apical hooks, and hypocotyls (Fig S2A) with a scalpel.  468 Dissected organs were then placed in pre-cooled 2ml safelock Eppendorf tubes containing two 1/8” steel  469 ball bearings (Weldtite, 3906141), and flash frozen in liquid nitrogen. Seeds were surface-sterilized in 0.05% Triton X-100 for  5 min, then in 1:1 household bleach:0.05% Triton X-100 for 10 min, and rinsed four times with sterile water.  Sterilized seeds were sown on 50 µM nylon mesh (Sefar BV) over half-strength MS medium (Duchefa) with  388 0.8% plant agar (Duchefa), pH 5.7. After 4 d stratification at 4 °C in darkness, seeds were exposed to ~125 μmol m⁻² s⁻¹ white light at 21 °C for 1 h , then wrapped in foil and grown vertically  in darkness at 21 °C for 23 h. Seedlings were subsequently transferred under safe green light to new 0.5×  MS plates with or without treatments (see below) and grown in darkness for 48 h unless otherwise stated.    For salt stress experiments, 50 mM or 75 mM of NaCl (Fluka), KCl (Amresco), NaNO3 (Sigma), KNO3 (Sigma),  and MgCl2 (Sigma) were added to 0.5 MS media. For osmotic stress experiments, 100 mM or 150 mM of  sorbitol was added to 0.5 MS media. Stock solutions of N-1-naphthylphthalamic acid (NPA; Supelco) were  made by dissolving directly in dimethyl sulfoxide (DMSO; Sigma) to reach a stock concentration of 10 mM,  which was later added to media to achieve a working concentration of 5 µM. Equal volumes of DMSO  (0.05%) were added to 0.5 MS as mock treatment. Each tube contained organs from 30- 470 45 seedlings, and were ground frozen for 30s at 25 Hz twice. RNA-isolation was carried out using the RNEasy  471 Plant Mini kit (Qiagen) with on-column DNAse treatment, in accordance with manufacturer instructions.  472 Three samples were combined during addition of RLT bumer, whereby the bumer was transferred from one  473 tube to the next, until the lysate of 90-125 seedlings was suspended in the bumer to be loaded into the 474 QIAshredder spin column Messenger RNA was purified from total RNA using poly-T oligo-attached magnetic beads. After fragmentation, the first strand cDNA was synthesized using random hexamer primers followed by the second strand cDNA synthesis. The library was ready after end repair, A-tailing, adapter ligation, size selection, amplification, and purification. 