The protocol of methylated RNA immunoprecipitation (MeRIP) was referred to Meng's study.54 Total RNA was extracted from stable METTL3 knockdown (shMETTL3) or METTL3 control (shNC) LoVo cells with Trizol reagent and then treated with DNase R (Qiagen). Chemically fragmented RNA (~100 nucleotides) was immunoprecipitated with anti‐m6A antibody (Abcam), washed three times with IP buffer (10 mmol/L Tris‐HCl, 150 mmol/L NaCl and 0.1% [vol/vol] Igepal CA‐630 [Sigma‐Aldrich]). RNA was eluted from the beads by incubating with elution buffer (IP buffer and 6.7 mmol/L N6‐methyladenosine salt [Sigma‐Aldrich]) for 1 hour at 4°C. Following ethanol precipitation, input and eluted RNA were then analysed by qRT‐PCR.
Anti m6a antibody
Manufactured by Abcam
Sourced in United States, United Kingdom
The Anti-m6A antibody is a research-use antibody that recognizes the N6-methyladenosine (m6A) modification in RNA. This antibody can be used to detect and study the distribution of m6A modifications in various RNA species.
Automatically generated - may contain errors
Lab products found in correlation
Magna rip kit, by Merck Group (6 mentions)
Amersham hybond n membrane, by GE Healthcare (6 mentions)
Rna fragmentation reagent, by Thermo Fisher Scientific (5 mentions)
Hybond n membrane, by GE Healthcare (4 mentions)
Bio dot apparatus, by Bio-Rad (4 mentions)
Protein a g magnetic bead, by Thermo Fisher Scientific (4 mentions)
Magna rip rna binding protein immunoprecipitation kit, by Merck Group (4 mentions)
Trizol reagent, by Thermo Fisher Scientific (4 mentions)
Dnase 1, by Merck Group (3 mentions)
Magnetic mrna isolation kit, by New England Biolabs (3 mentions)
Magna merip m6a kit, by Merck Group (3 mentions)
Ribominus eukaryote kit v2, by Thermo Fisher Scientific (3 mentions)
Rnase inhibitor, by Thermo Fisher Scientific (3 mentions)
Dynabead, by Thermo Fisher Scientific (2 mentions)
Fragmentation reagents kit, by Thermo Fisher Scientific (2 mentions)
Cap clip, by CellScript (2 mentions)
T4 pnk, by New England Biolabs (2 mentions)
Novaseq 6000, by Illumina (2 mentions)
Methylene blue, by Merck Group (2 mentions)
N6 methyladenosine 5 monophosphate sodium salt, by Merck Group (2 mentions)
65 protocols using anti m6a antibody
1
MeRIP Protocol for Analyzing m6A Methylation
2
KIAA1429 Interactome and m6A Profiling
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RIP assay was conducted as previously described.38 (link) Cell lysates were prepared with RIP lysis buffer (Magna RIP Kit; Millipore, USA) and then incubated with 5 μg of anti-KIAA1429 or rabbit IgG at 4°C overnight. The RNA-protein immunocomplexes were collected by protein A/G magnetic beads. After elution and purification, the purified RNA was analyzed by RT-PCR and qRT-PCR.
The m6A RIP was performed as previously described with some modifications.27 (link) Total RNAs were isolated from SUM-1315 stable KIAA1429/METTL3 knockdown and control cells and treated with DNase I (Sigma Aldrich, USA). RNAs were fragmented by RNA fragmentation reagents. Immunoprecipitations were performed using an anti-m6A antibody (1:1,000; Abcam, USA) previously bound to magnetic Dynabeads (Life Technologies, USA) in the RIP Immunoprecipitation Buffer (Magna RIP Kit; Millipore, MA, USA) and incubated with DNA-free fragmented RNAs. RNAs were extracted by miRNeasy Mini kit (QIAGEN, Germany) and subjected to qRT-PCR and normalized to input.
The m6A RIP was performed as previously described with some modifications.27 (link) Total RNAs were isolated from SUM-1315 stable KIAA1429/METTL3 knockdown and control cells and treated with DNase I (Sigma Aldrich, USA). RNAs were fragmented by RNA fragmentation reagents. Immunoprecipitations were performed using an anti-m6A antibody (1:1,000; Abcam, USA) previously bound to magnetic Dynabeads (Life Technologies, USA) in the RIP Immunoprecipitation Buffer (Magna RIP Kit; Millipore, MA, USA) and incubated with DNA-free fragmented RNAs. RNAs were extracted by miRNeasy Mini kit (QIAGEN, Germany) and subjected to qRT-PCR and normalized to input.
3
Quantifying m6A Methylation in mRNA
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mRNA from fresh tumors was isolated using Magnetic mRNA Isolation Kit (New England Biolabs, S1550S) and then denatured at 95°C for 3 min, followed by chilling on ice. Quantified mRNA was spotted on an Amersham Hybond‐N+ membrane (GE Healthcare, RPN3050B) and crosslinked to the membrane with UV radiation. The membrane was blocked in 5% of non‐fat milk PBST buffer and then incubated with anti‐m6A antibody (1: 2,000; abcam) overnight at 4°C. After incubating with HRP‐conjugated secondary antibodies, the membrane was visualized by SuperSignal West Femto Maximum Sensitivity Substrate (Thermo Fisher Scientific).
4
m6A Profiling of Bladder Cancer Cells
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For the m6A RNA binding experiments, the RNAs of bladder cancer cells stably transfected with either the lentiviral overexpression or control ALKBH5 vector were isolated and treated with RNase I (Sigma-Aldrich, USA). RNAs were fragmented by sonication for 10 s on an ice-water mixture. Immunoprecipitations were performed using an anti-m6A antibody (1:1,000; Abcam, USA) previously bound to magnetic Life Technologies Dynabeads (Thermo Fisher Scientific, USA) in the RIPA buffer (Magna RIP RNA-Binding Protein Immunoprecipitation Kit; Millipore Sigma, USA) and incubated with DNA-free fragmented RNAs. Beads were then treated with proteinase K (20 mg/mL) for 1.5 h at 42°C. Subsequently, RNAs were extracted using phenol/chloroform/isoamyl alcohol and subjected to qRT-PCR using primers for CK2α and normalized to input.
5
Methylated RNA Immunoprecipitation for MafA
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Methylated RNA immunoprecipitation coupled with qPCR (MeRIP-qPCR) was performed using the MeRIP kit (Bersinbio, Guangzhou, China), according to the manufacturer’s instructions. Briefly, total RNA was extracted from NIT-1 cells using TRIzol reagent, and the extracted RNA was fragmented using ultrasound treatment. The processed fragments were approximately 300 bp. After fragmentation, 50 μl of each RNA sample (the input sample) was stored at −80°C and the remaining portion of each RNA sample was immunoprecipitated with an anti-m6A antibody (Abcam, Cambridge, UK) or anti-IgG antibody. The RNA-antibody hybridization solution was incubated with Protein A/G magnetic beads for 1 h at 4°C in a vertical mixer. The beads were washed three times and digested with proteinase K at 55°C for 45 min. The supernatant was transferred to new RNase-free tubes, and the RNA was purified and subjected to qPCR. The MafA primer sequences were as follows:
Forward: 5′-CAGGAAAAGCGGTGCTGGAGG-3′,
Reverse: 5′-CGAAGCTCTGACCCCGGAAGG-3′.
Forward: 5′-CAGGAAAAGCGGTGCTGGAGG-3′,
Reverse: 5′-CGAAGCTCTGACCCCGGAAGG-3′.
6
mRNA Quantification using Anti-m6A Antibody
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mRNA was isolated from total RNA using the mRNA Purification Kit (TIANGEN) following the manufacturer’s instructions. mRNA was heated at 65°C for 15 minutes and cooled immediately on ice for 5 minutes. mRNA samples (500, 200, and 100 ng) were spotted on the Hybond-N+ membrane (GE Healthcare). After UV cross-linking, the membrane was washed with 1× TBST buffer and blocked with 5% skim milk. Then, the blot was incubated with the anti-m6A antibody (1:1,000, Abcam) overnight at 4°C. The membrane was washed, incubated with an anti-mouse antibody, and washed again. Finally, the membrane was exposed to Hyperfilm ECL, and images were acquired. Methylene blue interacted with mRNA and was used as the loading control.
7
Quantification of m6A RNA Methylation
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Briefly, equal amounts of RNA were denatured at 95°C for 5 min, followed immediately cooling at 4°C for 1 min. Afterward, RNA was added to Amersham Hybond N+ membranes and cross-linked for 30 s on a Stratalinker 2400 UV cross-linker with 2,000 mJ. The membranes were blocked with 5% skim milk for 1 hr at room temperature, and then the anti-m6A antibody (anti-m6A antibody, Abcam) was incubated overnight at 4°C. After several washes with 0.1% phosphate buffered saline with Tween-20, the membranes were incubated with horseradish peroxidase-coupled secondary antibody (7074s, Cell Signaling Technology) at room temperature for 1 hr. The membranes were developed with a Chemiluminescence Imaging System (Tanon 5200 SF, China).
8
m6Am-exo-seq and qPCR for Epitranscriptomic Analysis
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m6Am-exo-seq was performed according to protocols developed by the Shi group (24 (link)). Briefly, mRNA was extracted from PCIF1-KO and control Calu-3 cells using a Magnetic mRNA Isolation Kit. Aliquots (100 μg) of mRNA were fragmented using a Fragmentation Reagents Kit (Invitrogen, AM8740) according to the manufacturer’s protocol. Fragmented mRNAs were phosphorylated with T4 PNK (NEB, M0201S) and then dephosphorylated with Terminator 5′-Phosphate-Dependent Exonuclease (Lucigen). Finally, Cap-Clip (CellScript) was added to remove capped transcripts. Of the final uncapped fragmented mRNA preparation, 10% was reserved as input material, and the remaining 90% was subjected to immunoprecipitation with anti-m6A antibody (Abcam, ab151230). Immunoprecipitated RNA was eluted with RLT buffer (QIAGEN, 160051456), purified by ethanol precipitation, and prepared for library generation using a TruSeq mRNA library preparation kit (Illumina). Sequencing was performed at IGM Genomics Center, UCSD, using an Illumina NovaSeq 6000.
For m6Am-exo-qPCR, the same procedure as for m6Am-exo-seq was used through the anti-m6Am immunoprecipitation and RNA elution step. The eluted RNA and input samples were reverse transcribed and subjected to qPCR on a LightCycler 480 (Roche Diagnostics) using the primers listed inSI Appendix, Table S4 .
For m6Am-exo-qPCR, the same procedure as for m6Am-exo-seq was used through the anti-m6Am immunoprecipitation and RNA elution step. The eluted RNA and input samples were reverse transcribed and subjected to qPCR on a LightCycler 480 (Roche Diagnostics) using the primers listed in
9
Single-nucleotide-resolution Mapping of m6A in A498 Cells
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Single-nucleotide-resolution mapping of m6A of A498 cells was carried out according to previously published studies (Chen et al., 2015 (link); Linder et al., 2015 (link)) with some modifications. Briefly, the mRNA of A498 cells were purified by Dynabeads mRNA Purification Kit (Life Technologies) and fragmented to about 100 nt by the fragmentation reagent (Life Technologies). 2 μg of fragmented mRNAs were incubated with 5 μg of anti-m6A antibody (Abcam) in 300 μl immunoprecipitation buffer (50 mM Tris, pH 7.4, 100 mM NaCl, 0.05% NP-40) at 4°C for 2 h. The mixture was then irradiated three times with 0.15 Jcm–2 at 254 nm by a CL-1000 Ultraviolet Crosslinker (UVP), and then incubated with Dynabeads Protein A (Life Technologies) at 4°C for 2 h. After washing, end-repair and linker ligation, the enriched RNA were isolated from the beads by proteinase K digestion, and extracted by phenol–chloroform. Purified RNAs were reverse transcribed by Superscript III reverse transcriptase (Life Technologies). The cDNA from last step was with size selection on a 6% TBE-Urea gel (Life Technologies), and circularization and re-linearization by CircLigase II (Epicenter) and BamHI (NEB), respectively. Then the cDNA was amplified by AccuPrime SuperMix 1 enzyme (Life Technologies) for 20 cycles and sequenced by Illumina HiSeq X Ten according to the manufacturer’s instructions.
10
Quantifying m6A RNA Methylation
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Total RNA was extracted from PC9 cells and H3255 cells transfected with sh‐METTL3 or sh‐NC with TRIzol reagent (Thermo Fisher Scientific, USA). NanoDrop was used to test the concentration of RNA and dilute RNA to 100 ng/µL with RNase‐free water. 657 µL formamide (Sigma, USA), 210 µL 37% formaldehyde solution (Thermo Fisher Scientific, USA) and 133 µL MOPS 10X Solution (Thermo Fisher Scientific, USA) formulated into RNA incubation buffer. Add triple the volume of RNA incubation buffer to RNA solution and incubated at 65°C for 5 mins. Then, add same volume 20X Saline‐Sodium Citrate Solution (Sigma, USA) to solution mentioned above. 50ng, 100 ng, 200 ng and 400 ng RNA buffers were spotted onto a nylon membrane (Bio‐Rad, USA) by Bio‐dot apparatus (Bio‐Rad, USA). RNA was UV crosslinked to the nylon membrane. Then, the membrane was incubated with 0.02% Methylene blue (Sigma, USA) for 1 hour. The membrane was incubated with anti‐m6A antibody (1000 µg/mL, 1:1000 dilution, Abcam; catalog no. 208 577) for 12 hours at 4℃.
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