Rna clean and concentrator 5 column

For in vivo NAI-N₃ modification, the washed parasite pellets were subjected to in vivo NAI-N₃ modification procedures according to standards (Flynn et al., 2016 (link)) using 100 mM NAI-N₃ for 15 min as described previously (Qi et al., 2021 (link)) before total RNA extraction. Briefly, after lysis of RBCs by 0.05% saponin treatment and washing with PBS, the parasite pellet was resuspended in 200 µl of dimethyl sulfoxide (DMSO) solution or 100 mM NAI-N₃ solution, mixed by inversion and incubated at 37°C for 15 min. These modifications cause reverse transcription to stop one nucleotide before modification. During NAI-N₃ modification, we set a DMSO-treated negative control sample because the DMSO sample can provide an “input” sample. The reaction was stopped and collected at 14,000 g for 30 s at 4°C. Then, the pellet was resuspended in 10 ml of pre-warmed TRIzol, and total RNA was isolated followed by DNase treatment. For the icSHAPE in vitro libraries, heat-denatured total RNA or polyA-selected RNA samples from parasites were treated with DMSO solution at 95°C for 2 min and then transferred to ice for cooling. The time and concentration of modified NAI-N₃in vitro were 10 min at 37°C and 100 mM, respectively. Finally, the samples were transferred to ice to stop the reaction, and the RNA sample was purified by a Zymo RNA Clean and Concentrator-5 column.