title: In-vivo-SHAPE-MaP-Mock-NAI-replicate3
design: Arabidopsis seedlings were grown on 1/2 Murashige and Skoog (MS) plates containing 0.8% agar and 1% sucrose at 22 C under 12/12 h light/dark cycles with 55% relative humidity. For elf18 treatment, Arabidopsis seedlings were grown on plates for 7 days and transferred to liquid 1/2 MS solution and grown for one more day (8-day-old) before treated with 10 mM elf18 or water for one hour. Arabidopsis seedlings treated with elf18 or water were collected and immediately immersed in the fresh NAI solution (100 mM NAI) or in the DMSO solution as previously described. To enhance the permeability of NAI, samples immersed in the solution were vacuum infiltrated and incubate at room temperature for 20 min. To quench the reaction, DTT (dithiothreitol, Roche) was added to the solution for a final concentration of (0.5 M) and reacted for 2 min. The tissue was then immediately washed with water for 3 times, frozen in liquid nitrogen, ground, and subject to total RNA isolation using Direct-zol RNA Miniprep Plus Kit (Zymo). The purified RNA was then subject to DNase treatment by add 2 l Turbo DNase (2 U/l) and incubate for 30 min, followed by adding another 2 l Turbo DNase (2 U/l) and incubated for another 30 min. mRNAs were enriched twice through poly(A) selection using Oligo d(T)25 Magnetic Beads (NEB), and subject to reverse transcription. The cDNA product was cleaned up using Oligo Clean & Concentrator Kit (Zymo) and the library preparation was performed as described in Smola et al. under the randomer library preparation workflow. Agilent 2100 Bioanalyzer was used for the sample quality control. The 16 libraries were pooled at equal amount of DNA and subject to the next-generation sequencing using Illumina NovaSeq (S4, full flow cell) with pair-end 150 bp.