CAP-SHAPE-seq of WT Arabidopsis thaliana: biological replicate 2, technical replicate 2, with NAI
We modified the in vivo chemical probing protocol2 by changing the reagent from DMS to the SHAPE reagent: 2-methylnicotinic acid (NAI). The NAI was prepared as reported previously27. Briefly, five-day-old A. thaliana etiolated seedlings were suspended and completely covered in 20 ml 1XSHAPE reaction buffer (100mM KCl, 40mM HEPES (pH7.5) and 0.5mM MgCl2) in a 50 ml Falcon tube. NAI was added to a final concentration of 150mM and the tube swirled on a shaker (1000rpm) for 15min at room temperature (22C). This NAI concentration and reaction time had been optimized to allow NAI to penetrate plant cells and modify the RNA in vivo under single-hit kinetics conditions (Extended Data Fig. 1a). After quenching the reaction with freshly prepared dithiothreitol (DTT), the seedlings were washed with deionized water and immediately frozen with liquid nitrogen and ground into powder. Total RNA was extracted using RNeasy Plant Mini Kit (Qiagen) according to the manufacturer's instructions, followed by on-column DNaseI treatment in accordance with the manufacturers protocol. The control group was prepared using DMSO ((-)SHAPE) and following the same procedure described above.