Determination of site‐specific m6A modification levels using SELECT

SELECT protocol

This protocol is revised according to the previous article (Xiao et al, Angew Chem Int Ed Engl. 2018) ¹. 

• Always be RNAse-free!

Material and Reagents: 

10×CutSmart buffer (NEB, B6004V)

dTTP (ThermoFisher, R0171)

RNAse-Free H2O (ThermoFisher, AM9937)

Bst 2.0 DNA polymerase (NEB, M0537S)

SplintR ligase (NEB, M0375S)

ATP (NEB, P0756S)

Procedure:

1. Annealing primer design

1.1 The Up primer or Down primers of Annealing primer were designed as Figure 1 from Xiao et al, Angew Chem Int Ed Engl. 2018 ¹.

• An Example from Xia et al, EMBO Rep, 2021 ²：

Template sequence for m6A_modified Base 47 with yellow shadow from Xia et al, EMBO Rep, 2021 ²: 

attgcaccttgccggccacctttgctatctttgctgaagatgatggAcccaaactcgacttctgaagatgtaaaatttacacctgacccataccaggtgccttttgtacaagcttttgaccaagctaccagagtctatcaggacctgggagggccatcgcaagctcctttgccttgtgtgctgtggccggtgctgccagagcctctgccacaaggccagctaactgcctatcatgtttcaaccgctccgactgggtcgtggttttctgcccctcagcctgctcctgagaatgcttatcaagcttatgcagcacctcagctgttcccagtctccgacataacccagaatcaacagactaaccaagccgggggagaagcacctcaacctggagacaattctactgttcaaacagcagcagcagtggtgtttgcttgccccggggctaaccaaggacaacagctagcagacattggtgttccacagcctgcaccagtggctgccccggcacgacgcacacggaaaccacaacagccagaatcg

1.2 The Up primer or Down primers were designed with 20-21 bp PCR adapter (shown in lower cases) and 20-30 bp annealing oligo (shown in upper cases). 

The Annealing primer for Base 47 is designed as bellow:

The Up primer: tagccagtaccgtagtgcgtgTTACATCTTCAGAAGTCGAGTTTGGG

The Down primer: 5phos/CCATCATCTTCAGCAAAGATAGCAAAGcagaggctgagtcgctgcat

1.3 The SELTCT qPCR primer is designed according to the PCR adapter. 

qPCRF primer: ATGCAGCGACTCAGCCTCTG

qPCRR primer: TAGCCAGTACCGTAGTGCGTG

2. Annealing, elongation, and ligation

2.1 Prepare Annealing mixture:

             3ug Total RNA

             40 nM Up Primer

             40 nM Down Primer

             5 μM dTTP

             10×CutSmart buffer （2ul）

             RNAse-Free H2O

             Total to 17 ul

2.2 The RNA and primers were annealed by incubating mixture at a temperature gradient: 

             90°C for 1min,

             80°C for 1min,

             70°C for 1min,

             60°C for 1min,

             50°C for 1min,

             40°C for 6min,

             4°C, hold.

2.3 Subsequently, a 3 μl of mixture containing 0.01 U Bst 2.0 DNA polymerase (NEB), 0.5 U SplintR ligase (NEB) and 10 nmol ATP (NEB) was added in the Annealing mixture to the final volume 20 μl of Final reaction mixture.

2.4 The Final reaction mixture is incubated at:

             40°C for 20 min, 

             80°C for 20 min,

             4°C, hold.

3. SELECT qPCR step

3.1 Prepare qPCR mixture:

             2× PowerUp™ SYBR™ Green Master Mix (Applied Biosystems) (10 ul),

             200 nM qPCRF primer,

             200 nM qPCRR primer,

             2μl of the Final reaction mixture

             ddH2O

             Total to 20 ul

3.2 qPCR was run in Applied Biosystems ViiA^™ 7 Real-Time PCR System (Applied Biosystems, USA) or StepOnePlus™ Real-Time PCR System (Applied Biosystems).

qPCR is run at the following condition:

             95°C, 5min; 

             (95°C, 10s; 60°C, 35s)×40 cycles; 

             95°C, 15s;

             60°C, 1min;

             95°C, 15s (collect fluorescence at a ramping rate of 0.05°C/s);

             4°C, hold.

3.3 The relative m6A level is determined by calculating the 2^-ΔCt of the targeted m6A base relative to the control A base.

1.     Xiao Y, Wang Y, Tang Q, Wei L, Zhang X, Jia G. An Elongation- and Ligation-Based qPCR Amplification Method for the Radiolabeling-Free Detection of Locus-Specific N(6) -Methyladenosine Modification. Angew Chem Int Ed Engl. Dec 3 2018;57(49):15995-16000. doi:10.1002/anie.201807942

2.     Xia TL, Li X, Wang X, et al. N(6)-methyladenosine-binding protein YTHDF1 suppresses EBV replication and promotes EBV RNA decay. EMBO Rep. Apr 7 2021;22(4):e50128. doi:10.15252/embr.202050128