RNAseq of 3 day old M. truncatula seedling roots treated with 1 uM MtPIP1 (RLPSG(Hyp)SPKGPGH) peptide. Each replicate comprises 20-40 seedling roots.
Overnight germinated M. truncatula seedlings were grown vertically on 1% water agarose medium (Life Technologies Ultrapure Agarose). Seedlings were transferred onto sterile media and placed between filter papers  (Whatman; grade 0858, reference no. 10 334 365) overlaying the gel medium. Plates were wrapped in black plastic and kept in a growth chamber at 22C with a 16-h-day/8-h-night cycle at approximately 120 mol photons m2 s1 for 10 d. On the third day, seedlings were carefully removed from plates and equal sized plants were transferred to glass beakers containing 1 uM of the appropriate peptide in water at pH 6.8. The bottom half of the beaker was covered in black plastic and the beaker placed in a growth chamber at 22C. After three hours, seedling roots were excised using a scalpel blade, 20-40 seedling roots pooled and immediately frozen in liquid Nitrogen. RNA was extracted using a Qiagen kit according to Manufacturer's protocol.