Sequencing libraries were prepared as outlined in Fig. 1 with a modified version of the protocol used for ribosome profiling32 (link). Specifically, DMS treated mRNA samples were denatured for 2 min at 95°C and fragmented at 95°C for 2 min in 1X RNA fragmentation buffer (Zn2+ based, Ambion). The reaction was stopped by adding 1/10 volume of 10X Stop solution (Ambion) and quickly placed on ice. The fragmented RNA was run on a 10% TBU (Tris Borate Urea) gel for 60 min. Fragments of 60–80 nucleotides in size were visualized by blue light (Invitrogen) and excised. Gel extraction was performed by crushing the purified gel piece and incubating in 300 µl DEPC treated water at 70°C for 10 min with vigorous shaking. The RNA was then precipitated by adding 33 µl NaOAc, 2 µl GlycoBlue (Invitrogen), and 900 µl 100% EtOH, incubating on dry ice for 20 min and spinning for 30 min at 4°C. The samples were then re-suspended in 7 µl 1X PNK buffer (NEB) and the 3’phospates left after random fragmentation were resolved by adding 2 µl T4 PNK (NEB), 1 µl of Superase Inhibitor (Ambion) and incubating at 37°C for 1h. The samples were then directly ligated to 1 µg of microRNA cloning linker-1, /5rApp/CTGTAGGCACCATCAAT/3ddC/ (IDT DNA) by adding 2µl T4 RNA ligase2, truncated K227Q (NEB), 1 µl 0.1M DTT, 6 µl 50%PEG, 1 µl 10X ligase2 buffer, and incubating at room temperature for 1.5 hr. Ligated products were run on a 10% TBU gel for 40 min, visualized by blue light, and separated from unligated excess linker-1 by gel extraction as described above. Reverse transcription (RT) was performed in 20 µl volume at 52°C using SuperscriptIII (Invitrogen), and truncated RT products of 25–50 nucleotides (above the size of the RT primer) were extracted by gel purification. The samples were then circularized using circ ligase (Epicenter), and Illumina sequencing adapters were introduced by 8–10 cycles of PCR.