This is a protocol preprint. Preprints are preliminary reports that have not undergone peer review. They should not be considered conclusive, used to inform clinical practice, or referenced by the media as validated information.
Method Article
Nm-seq
Dan Dominissini
Dominissini Lab (Sheba, Tel-Aviv)
Corresponding Author
License:
This work is licensed under a Creative Commons Attribution 4.0 International License. Read Full License
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).
Keywords
2-O-methylation, RNA, Nm, epitranscriptome, modifications, 2prime-O-methylation
Figure 1
Figure 2
Figure 3
Figure 4
• 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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Associated Publications
Method Article
Nm-seq
Dan Dominissini
Dominissini Lab (Sheba, Tel-Aviv)
Corresponding Author
Version History
CURRENT STATUS: Posted
Version 1
Posted 15 May, 2017
Metrics
Comments: 0
HTML Views: ...
License:
This work is licensed under a Creative Commons Attribution 4.0 International License. Read Full License
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).
Figure 1
Figure 2
Figure 3
Figure 4
• 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.