M6a rna enrichment kit

Total RNA was extracted by RNAiso Plus (Takara, Japan), and DNAse was added to remove DNA. MeRIP assays were performed using the m⁶A RNA Enrichment Kit (Epigentek, USA) according to the manufacturer’s protocols. Target m⁶A-containing fragments were pulled down using a beads-bound m⁶A capture antibody, and RNA sequences in both ends of the m⁶A-containing regions were cleaved using Cleavage Enzyme Mix. The enriched RNA was then released, purified and eluted. QRT-PCR was performed following MeRIP to quantify changes in target gene m⁶A methylation.

MeRIP assays were performed using the m⁶A RNA Enrichment Kit (Epigentek, USA). Target m⁶A-containing fragments were immunoprecipitated using a bead-bound m⁶A capture antibody, and RNA sequences at both ends of the m⁶A-containing regions were cleaved using Cleavage Enzyme Mix. The enriched RNA was released, purified, and eluted. Quantitative reverse transcriptase-PCR (qRT-PCR) analysis was performed following MeRIP to quantify changes in target-gene m⁶A modification. Primers (Table S1) were synthesized by Sangon Biotech Co., Ltd. (Shanghai, China).

After extracting the total RNA (20 μg) of SW480 cells, the m⁶A RNA Enrichment Kit (EpiGentek, NY, USA) was used to enrich and purify the methylated RNA according to the manufacturer’s instructions. Finally, RNA reverse transcription PCR and qRT-PCR were performed to detect the m⁶A sites. The primer sequences for the m⁶A site are listed in Supplemental Table S1.

Total RNA extraction was conducted by using the RNAiso Plus (Takara, Shiga, Japan), after which DNase was added to remove DNA. An m6A RNA enrichment kit (EpigenTek) was used to detect MeRIP levels according to the manufacturer’s instructions. The m6A-containing target fragment was pulled down using a bead-bound m6A capture Ab, after which the RNA sequence containing both ends of the m6A region was cleaved using a lyase cocktail. The enriched RNA was released, purified, and eluted. Quantitative real-time PCR (QRT-PCR) was performed after MeRIP to quantify changes in target gene m6A methylation.
Antibodies against ALKBH5 (Abcam, #ab195377) were used in RNA-binding protein immunoprecipitation (RIP) assays. RIP assays were performed using an EZ-Magna RIP™ RNA-Binding Protein Immunoprecipitation Kit (Millipore, Billerica, MA, USA) according to the manufacturer’s instructions. Cells at approximately 90% confluence were lysed using complete RIP lysis buffer containing RNase Inhibitor (Millipore) and protease inhibitor, and 100 μl of whole-cell extract was subsequently incubated with RIP buffer containing magnetic beads conjugated to specific antibodies. The detailed method of the RIP assay was described in a previously published study^{12 (link)}.

cDNA was synthesized from the 1ug mRNA with HiScript II Reverse Transcriptase (Vazyme, R232). Real-time PCR was performed with Universal SYBR qPCR Master Mix (Vazyme, Q511). β-ACTIN gene was used for the internal control and the 2^{^(−ΔΔCt)} methods were used to calculate the relative level of each mRNA. The sequence of PCR primers is shown in Supplementary Table S1.
The m6A RNA Enrichment Kit (Epigentek, Farmingdale, IL, USA) was applied to perform the MeRIP assays in line with the manufacturer’s protocols. The 10ug total RNA were input and 2 ug m6A antibody were immunoprecipitated in the MeRIP-qPCR, which is consistent with MeRIP-seq. HiScript II Reverse Transcriptase (Vazyme, R232) and AceQ^® Universal SYBR qPCR Master Mix (Vazyme, Q511) were, respectively, applied to perform reverse transcription and real-time qPCR. The primers of MeRIP-qPCR were designed according to the identified m6A peak in differential peak analysis. The mRNA of the corresponding sample without m6A immunoprecipitation was used as the internal control. Additionally, the 2^{^(−ΔΔCt)} methods were used to calculate the relative level of each mRNA. Primer sequences are shown in Supplementary Table S1.