Biological replicate 1 with 2.5% DMS treatment by Structure-seq in vivo in Oryza sativa
5-day-old etiolated rice seedlings were thoroughly covered in 1×DMS reaction buffer with 40mM HEPES (pH7.5), 100mM KCl and 0.5mM MgCl2. Dimethyl sulfate (DMS) was added to a final concentration of 2.5% with vigorous vortex until dissolved completely. Seedlings were treated for 15min at 28°C. Dithiothreitol (DTT) was added to a final concentration of 0.5M with vigorous vortex until dissolved completely to quench DMS. The seedlings were washed by deionized water. Then total RNA was extracted and purified with RNeasy Plant Mini Kit (Qiagen). Structure-seq library construction method published in 2014 was applied in the further experiment. We performed two rounds of poly(A) selection using the Poly(A) Purist MAG Kit (Ambion) and started library construction with 500ng poly(A)-selected RNAs. After reverse transcription by random hexamers fused with Illumina TruSeq adapter, ligation with 5’ssDNA linker by CircLigase (Epicentre) and cDNA size selection (>100nt), PCR amplification was performed using Illumina TruSeq Primers. PCR products were selected between 200bp and 650bp. The dsDNA library was subjected to 50bp single-end sequencing on Illumina HiSeq 4000 platform.
NpDMS_1_31_1