We used the SHAPE reagent, 2 methylnicotinic acid to do the in vivo RNA secondary structure chemical probing. NAI was prepared as reported previously. Briefly, Arabidopsis thaliana seedlings were completely covered in 20 mL SHAPE reaction buffer (100 mM KCl, 40 mM HEPES (pH7.5) and 0.5 mM MgCl2) in a 50 mL Falcon tube. NAI was added to a final concentration of 1 M and the tube swirled on a shaker (1000 rpm) for 15 min at room temperature (22 celsius) or 30 min at 4 Celsius. This high NAI concentration allows NAI to penetrate plant cells and modify the RNA in vivo. 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 hot phenol method, followed by DNaseI treatment in accordance with the manufacturer's protocol. The control group was prepared using DMSO (dimethyl sulfoxide, labeled as minus SHAPE), following the same procedure as described above. 2ug plus SHAPE or minus SHAPE RNA samples was added into 19 uL buffer system containing with 2 uL 0.5 uM RNA-DNA hybrid adaptors, 4 uL reaction buffer, and 1uL TGIRT-III III enzyme.The reaction system was pre-incubated at room temperature for 30 minutes, then 1 uL of 25 mM dNTPs (an equimolar mixture of dATP, dCTP, dGTP, and dTTP at 25 mM each RNA grade) added. The whole reaction system in the tube was incubated at 60 celsius for 120 minutes. To remove the TGIRT-III enzyme from the template, 1 uL of 5 M NaOH was added and incubated at 95 celsius for 3 minutes. The sample was cooled down to room temperature and neutralized with 1 uL of 5 M HCl before the clean-up of the cDNAs with a MinElute Reaction Cleanup Kit. To capture Class I and Class II COOLAIRs along with 18S rRNA, PCR reactions with 10 cycles were conducted with specific primers using KOD Xtreme Hot Start DNA Polymerase (Novagen). The amplified DNA fragments from the eight replicates of PCR reactions were merged for achieving sufficient DNAs. The resulting DNAs were size-selected by Solid Phase Reversible Immobilization (SPRI) size selection system (BECKMAN COULTER). Two independent biological replicates were generated for both plus SHAPE and minus SHAPE smStructure-seq libraries. The purified DNA samples were subjected to PacBio library construction by BGI company using PacBio Sequel 3.0. For HIV-1 RRE61 and tenA treatment, the details are in the manuscript.