In vivo genome-wide profiling of RNA secondary structure reveals novel regulatory features-(+)DMS_Structure_Seq_Biological_replicate_II
Five day-old Arabidopsis thaliana etiolated seedlings were suspended intact and completely covered in 20 mL 1X DMS reaction buffer in a 50 mL Falcon tube that contained 100 mM KCl, 40 mM HEPES (pH 7.5), and 0.5 mM MgCl2. DMS was added to a final concentration of 0.75% (~75 mM) and allowed to react for 15 min at room temperature with periodic swirling. To quench the reaction, freshly-prepared dithiothreitol (DTT) was added to a final concentration of 0.5 M, and after swirling for 2 min the reaction mixture was decanted and the seedlings were washed with ~2 x 50 mL of deionized water. The seedlings were immediately frozen with liquid N2 and subjected to total RNA extraction, following the protocol described in the RNeasy Plant Mini Kit (Qiagen). In vivo total RNA isolation was followed by one round of poly(A) selection using the Poly(A) purist Kit (Ambion). The poly(A) RNA (2 µg) was then treated with TURBO DNase (Ambion) following the manufacturer’s protocol, followed by phenol chloroform extraction and ethanol precipitation. Then the poly(A) RNA was resuspended in RNase-free water and subjected to reverse transcription using the SuperScript™ III First Strand Kit (Invitrogen) and random hexamers fused with Illumina TruSeq Adapter. The resultant first strand cDNAs were then ligated at their 3’-ends to a single-stranded (ss) DNA linker using CircLigase™ ssDNA Ligase (Epicentre). PCR amplification was performed on the ligated cDNA using Illumina TruSeq Primers. The resulting double-stranded (ds) DNA libraries were subjected to next generation sequencing on an Illumina HiSeq 2000.