Arabidopsis_vivo_SHALiPE_replicate2
5-day-old etiolated Arabidopsis seedlings were thoroughly covered in 1SHAPE reaction buffer with 40mM HEPES (pH7.5), 100mM KCl and 0.5mM MgCl2. Seedlings were treated at 500rpm for 15min under 22C. After treatment, total RNA was extracted and purified with RNeasy Plant Mini Kit (Qiagen). RNA with poly-A tail was enriched by two rounds of poly(A) selection using the Poly(A) Purist MAG Kit (Ambion). Poly-A selected RNA was applied to reverse transcription with home-made RT buffer (20 mM Tris (pH 8.3), 100 mM LiCl, 3 mM MgCl2, 1 mM DTT) and random hexamers. The 3end of resultant cDNAs were ligated to a ssDNA linker using Circligase ssDNA Ligase, ligated cDNA was purified by Urea-PAGE. PCR amplification was performed using Illumina TruSeq Primers and KAPA Library Amplification Kits. PCR products between 200bp to 650bp were purified using agrose gel and subjected to paired-end sequencing on Illumina HiSeq 4000 platform.