In-vitro-SHAPE-MaP-DMSO-replicate2
Arabidopsis seedlings treated in mock condition were collected, frozen in liquid nitrogen, ground, and subject to total RNA isolation using Direct-zol RNA Miniprep Plus Kit (Zymo). The purified RNA was subject to DNase treatment by adding 2 l Turbo DNase (2 U/l) and incubated in 37 C for 30 min, followed by addition of another 2 l Turbo DNase (2 U/l) and incubated for another 30 min. RNA was then purified by RNA Clean & Concentrator Kit (Zymo). mRNA was enriched twice through poly(A) selection using Oligo d(T)25 Magnetic Beads (NEB). To probe the in vitro RNA secondary structures, 500 ng purified mRNA was mixed with NAI (100 mM) or DMSO in a SHAPE reaction buffer (100 mM HEPES, 6 mM MgCl2 and 100 mM NaCl) and incubated at room temperature for 5 min. The reaction was then quenched by purifying RNA using RNA Clean & Concentrator Kit (Zymo). The treated mRNA was then 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. Libraries were pooled and subject to the next-generation sequencing using Illumina NovaSeq with pair-end 150 bp.