Oligo clean concentrator kit

The isolated small RNAs’ other domain modifications were removed by AlkB and AlkB-mut, which quenched with 0.5 mol/L EDTA to a final concentration of 5 mmol/L EDTA. Next the AlkB-treated RNAs were recovered by Oligo Clean & Concentrator kit (ZYMO RESEARCH, D4060) and divided into three groups: NaCNBH₃ (treated with NaCNBH₃ and without pre-treatment of mild alkali), Deacetylation (treated with NaCNBH₃ and pre-treatment of mild alkali), Mock (without any treatment). Then the RNAs were precipitated with 2.5 volume cold ethanol at −20 °C for at least four hours and desalted with 75% cold ethanol. After precipitation, the RNAs were ligated with 3′ adapters, hybridized with reverse transcription primers and ligated with 5′ adapters. Ligated RNAs were reverse transcribed using TGIRT-III (InGex, TGIRT50) and performed PCR amplification. Subsequently, the products were purified by QIAquick PCR Purification kit (QIAGEN, 28104) and circularized by the splint oligo sequence forming the single strand circle DNA, followed by sequencing with MGISEQ-2000 (Fig. 5a). For total RNA reduction and misincorporation sequencing, we fragmented RNA and skipped the step of small RNA isolation. We then carried out the remaining steps as described in the TRMC-seq (Fig. 5a).

sAvL1-451 DNA were 5′-end-labeled with [γ−32P]dATP (PerkinElmer) using T4 polynucleotide kinase (NEB) and purified from excess of radioactive nucleotides using Oligo Clean & Concentrator kit (Zymo Research) following the manufacturer’s protocols. Binding reactions were set up in 10 µl total volume in a buffer with final concentrations 100 mM KCl, 10 mM Tris, pH7.4, 0.1 mM EDTA, 0.1 mM DTT, supplied with 500 ng LightShift^™ Poly (dI-dC) (Thermo Scientific). Addition of 2.5 µl of AvMBD proteins provided 5% glycerol per reaction. Proteins were first pre-incubated with non-radioactive DNA for 15 min at RT. Then, ³²P-labeled DNA was added to a final concentration of 0.05 nM, and reactions were incubated for additional 30 min at RT. After supplying with 6× EMSA gel-loading solution (Thermo Scientific), samples were loaded onto 6% DNA Retardation gels. Samples were run at 90 V in 0.5× TBE buffer (44.5 mM Tris–HCl, pH 8.3, 44.5 mM boric acid and 1 mM EDTA) at 4 °C for 90 min. Gels were dried using Model 583 Gel Dryer (BioRad), exposed with phosphorimaging plate (Fujifilm), scanned on Typhoon FLA 7000, and analyzed using Image Quant TL v8.1 software.

DM-tRNA-seq was performed following the previously reported protocol [27] (link), [47] (link) with some modifications. Small RNAs (< 200 nt) were first purified using the Quick-RNA Microprep kit (Catalog No. R1050, Zymo Research, Orange, CA). Isolated small RNAs were treated with recombinant wild-type and D135S AlkB proteins to remove the dominant methylations on RNAs. Then, demethylated RNAs were purified with Oligo Clean & Concentrator kit (Catalog No. D4060, Zymo Research). After that, AlkB-treated RNA libraries were constructed with NEBNext Small RNA Library Prep Set (Catalog No. E7330S, New England Biolabs, Ipswich, MA). The cDNA libraries were sequenced on Illumina HiSeq X10 with paired-end 2 × 150 bp read length.

10 µL of 10 µM RNA template-loop-primers (5’-UUUUCAUCGCGUAGUUUUCUACGCG-3’ for RDV-TP extension) in 1 × RdRp reaction buffer was annealed by heating to 75 °C for 3 min and cooling to room temperature. 5 µL of 8 µM RdRp complex (nsp12/nsp7/nsp8)^{38 (link)} in 1 × reaction buffer was added to the annealed RNA template-loop-primer solution and incubated for an additional 10 min at room temperature. Finally, 5 µL of a solution containing 0.2 mM RDV-TP in 1 × reaction buffer was added and incubation was carried out for 2 h at 30 °C. The final concentrations of reagents in the 20 µL extension reactions were 2 µM nsp12/nsp7/nsp8, 5 µM RNA template-loop-primer, and 50 µM RDV-TP. The 1 × reaction buffer contains the following reagents: 10 mM Tris–HCl pH 8, 10 mM KCl, 2 mM MgCl₂ and 1 mM β-mercaptoethanol. Desalting of the reaction mixture was performed with an Oligo Clean & Concentrator kit (Zymo Research) resulting in ~ 10 µL purified aqueous RNA solutions. 1 µL of each solution was subjected to MALDI-TOF MS (Bruker ultrafleXtreme) analysis. The remaining ~ 9 µL extended template-loop-primer solutions were used to test exonuclease activity as described below.

Total RNA of cells was fragmented in Fragmentation Reagent (Ambion) at 94°C for 5 min. And then the fragmented RNA was purified according to the instruction of Oligo Clean & Concentrator kit (ZYMO RESEARCH). About 100 ng fragmented RNA was used to construct the input library, and the rest was used for immunoprecipitation. A total of 25 μl of Pierce™ Protein A/G Magnetic Beads (Thermo Fisher Scientific) and 6 ul of 0.5 μg/μl anti-m⁶A primary antibody (202003, Synaptic Systems) were mixed in 1×IP buffer and incubated for 3 h at 4 °C in advance. And then the mixture was incubated with the fragmented mRNA which was prepared for immunoprecipitation overnight at 4 °C. After 5 times of strict washing, the captured RNA was eluted by competition with N6-methyladenosine (Sigma-Aldrich).