In vivo wheat RNA structure landscape reveals RNA structure-mediated regulation of subgenomic-dominant translation

5 day old etiolated seedlings of wheat Kronos were applied to SHAPE structrome-seq, polysome-seq or RNA-seq respectively. Structrome-seq, 5 day old etiolated Kronos seedlings were harvested by carefully removing the remaining seed, and incubated with 200 mM NAI at 22 degree for 15 minutes. To generate a control without NAI probing, a same volume of DMSO to that of NAI was added into the incubation buffer. The washed seedlings were then harvested and ground into fine powder in liquid nitrogen and applied to RNA extraction. PolyA-selected RNA was recovered and reverse transcribed. cDNA ligation was performed using Circligase ssDNA Ligase to ligate the 3 end of cDNA to a ssDNA linker for 12 hrs at 65 degree. Ligation product over 100 nt was recovered after gel separation. Purified ligated cDNA was amplified. The PCR products were purified using agarose gel to recover the fragments of 200-650 bp and sequenced. Polysome-seq, Around 500mg wheat seedlings were harvested and grounded into fine powder in liquid nitrogen. Precooled polysome extraction buffer was added and incubated on ice for 30 minutes for full lysis. The lysate was applied to centrifugation with 13200 rpm for 15 min at 4 degree, supernatant was transferred to sucrose gradient with salt buffer. Polysomes were fragmented by 4 hours of centrifugation at 40000 rpm. Low to high sucrose gradient was collected, RNA was extracted from the fractions corresponding to the polysomes and applied to library generation. RNA-seq, Total RNA was extracted from the wheat seedlings, two rounds of polyA selection were performed, RNA-seq libraries were generated and sequenced.