The ribose of RNA nucleotides can be 2'-O-methylated (Nm). Despite advances in high-throughput detection, its inert chemical nature still limits sensitivity and precludes mapping in messenger RNA (mRNA). We leveraged the differential reactivity of 2'-O-methylated and 2'-hydroxylated nucleosides to periodate oxidation to develop Nm-seq, a sensitive method for transcriptome-wide mapping of Nm with base precision. This protocol accompanies Dai et al, Nature Methods, published online May 15, 2017 (10.1038/nmeth.4294).
Method Article
Nm-seq
https://doi.org/10.1038/protex.2017.088
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2-O-methylation
RNA
Nm
epitranscriptome
modifications
2prime-O-methylation
• Total RNA, DNase-treated
• Molecular biology grade, RNase-free water (Biological Industries, cat. no. 01-866-1B)
• Sodium periodate (Sigma-Aldrich, cat. No. 311448)
• L-Lysine monohydrochloride (Sigma-Aldrich, cat. no. 62929)
• Ethylene glycol (Sigma-Aldrich, cat. no. 03747)
• RNA Fragmentation Reagents: Fragmentation Reagent (10X) and Stop Solution (Thermo Fischer Scientific, cat. no. AM8740)
• RNA Clean & Concentrator (Zymo Research, cat. no. R1015)
• Shrimp Alkaline Phosphatase and CutSmart Buffer (10X) (New England Biolabs, cat. no. M0371S)
• Antarctic Phosphatase and Reaction Buffer (10X) (New England Biolabs, cat. no. M0289S)
• T4 Polynucleotide Kinase (3' phosphatase minus) and Reaction Buffer (10X) (New England Biolabs, cat. no. M0236S)
• Adenosine 5'-Triphosphate (New England Biolabs, cat. no. P0756S)
• NEBNext® Small RNA Library Prep Set for Illumina (New England Biolabs, cat. no. E7330S)
• Light-protected tubes
REAGENT SETUP
Lysine-HCl buffer, 2 M, pH 8.5: Dissolve 3.653 g of L-Lysine monohydrochloride in 10 ml of molecular biology grade, RNase-free water. Titrate to pH 8.5 with Sodium hydroxide.
Sodium periodate solution, 200 mM: Dissolve 42.778 mg of sodium periodate in 1 ml of molecular biology grade, RNase-free water. Protect from light.
Standard molecular biology lab equipment
RNA FRAGMENTATION
1| Set up the following fragmentation reaction in a thin-walled 200 μl PCR tube. Mix well by vortex and spin down.
2| Incubate at 95 °C for 5 min in a pre-heated thermal cycler block with the heated lid closed. Remove tubes from block and immediately add 2 μl of stop solution. Mix well by vortex and spin down, and place on ice.
3| Purify fragmented RNA using RNA Clean & Concentraton-5 kit, according to the manufacturer’s instructions. Elute in 15 μl molecular biology grade, RNase-free water. Note that each RNA Clean & Concentrator-5 column is suitable for purification of up to 10 μg RNA.
3’ END REPAIR
4|Set up the following 3’ end repair reaction in a thin-walled 200 μl PCR tube. Mix well by gentle tapping and spin down.
5| Incubate at 37°C for 30 minutes.
6| Purify 3’ end-repaired fragmented RNA using RNA Clean & Concentraton-5 kit, according to the manufacturer’s instructions. Elute in 15 μl molecular biology grade, RNase-free water.
OXIDATION-ELIMINATION-DEPHOSPHORYLATION CYCLES
7|Set up the following oxidation-elimination reaction in a dark tube. Mix well by vortex and spin down.
8| Incubate at 37 °C for 30 minutes with shaking.
9| Quench the reaction by adding 2 μl of ethylene glycol. Mix well and spin down.
10| Dephosphorylate by adding 5 μl of CutSmart Buffer (10X), 2 μl of rSAP enzyme (2 units) and 1 μl of molecular biology grade, RNase-free water (total reaction volume of 50 μl), mix well and spin down.
11| Incubate at 37 °C for 30 minutes with shaking.
12| Purify RNA using RNA Clean & Concentrator-5 kit, according to the manufacturer’s instructions. Elute in 32 μl molecular biology grade, RNase-free water.
13| Repeat steps 7-12 seven more times.
FINAL OXIDATION-ELIMINATION
14| Repeat steps 7-9 once. Make sure not perform dephosphorylation (steps 10-11).
15| Purify RNA using RNA Clean & Concentraton-5 kit, according to the manufacturer’s instructions. Elute in 32 μl molecular biology grade, RNase-free water.
5’ PHOSPHORYLATION
16|Set up the following 5’ dephosphorylation reaction. Mix well by gentle tapping and spin down.
17| Incubate at 37 °C for 30 minutes.
18| Purify RNA using RNA Clean & Concentraton-5 kit, according to the manufacturer’s instructions. Elute in 8 μl molecular biology grade, RNase-free water.
LIBRARY PREPARATION FOR MASSIVELY PARALLEL SEQUENCING
19| Use RNA from step 18 to construct a small RNA library using NEBNext® Small RNA Library Prep Set for Illumina, according to the manufacturer’s instruction, with the exception that 3’ adaptor ligation should be performed overnight at 16 °C.
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