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MeRIP Sequencing Q&A

Inquiry

  • General Questions

  • Can meRIP-seq study m6A modifications of RNA?
    • Absolutely. Currently, m6A modifications in RNA are mainly studied by MeRIP-seq.
  • Can m6A be analyzed in association with circ RNA?
    • Yes, we can. We can build a library for circ RNA alone, to collect 5 ug of circle RNA for enrichment.
  • Is it true that m6A-seq does not need to be greater than 3 samples per group, and is it okay to do m6A without duplicates and only once per sample?
    • Except for cell line samples, which can be done with only 2 replicates (usually for two different shRNA or siRNA methods), usually even primary cells are three biological replicates.
  • Why are the genes of chloroplast and mitochondria compared in MeRIP data?
    • MeRIP library construction is based on the amount of mRNA to determine the type of library construction. If chloroplast and mitochondrial genes are matched in MeRIP data, it is probably a problem of extraction, there is no good way to remove mitochondria and chloroplasts, the only way is to increase the sequencing volume to increase the valid data.
  • Are MeRIP and RIP similar?
    • MeRIP (also known as m6A-IP) and RIP are different in that they have different experimental purposes: the former detects the m6A level of a gene, while the latter detects the mRNA bound to a reading protein. The m6A antibody can theoretically bind any RNA with m6A modification with 99% specificity and there is no non-specific enrichment.
  • How to design MeRIP-seq experiments?
    • • Samples: Cells, tissues, total RNA after extraction (limited to species with reference genome).
      • Grouping: Cellular model: experimental group and control group, 3:3 recommended
      • Tissues: normal group and disease group, 5:5 recommended
      • Intra-group control: IP group and input group
  • Do I need to interrupt after RNA enrichment in MeRIP-seq?
    • It is possible to interrupt or not to interrupt. If you want to interrupt, please make sure that the target is only mRNA or lncRNA before interrupting. If it is only mRNA, you can use oligo dT beads to enrich it first and then interrupt it. If both lncRNA and mRNA are to be studied, the rRNA of total RNA should be digested with the kit before interruption.
  • How to elute the RNA from m6A antibody if my RNA volume is low
    • If the RNA starting amount is low, the elution volume (elution buffer) can be increased by 25-30% and the number of elutions can be increased from 2 to 4-6 times.
  • How to extract the RNA after IP?
    • RNA from IP can be extracted by using phenol:chloroform:isoamyl alcohol with a volume ratio of 25:24:1, or by adding Trizol reagent directly.
  • What is your MeRIP-seq library building and sequencing process?
    • Each MeRIP-seq set consists of two types of samples, the immunoprecipitation (IP) sample and the Input control sample. The RNA molecules are first fragmented into 100-200nt fragments. The IP sample provides an unbiased measurement of the methylated RNA fragment by means of an anti-m6A specific antibody. Meanwhile, the Input control sample reflects the abundance of the base RNA. The localization of the whole transcriptome m6A is given by library building, high-throughput sequencing and bioinformatics analysis.
  • What MeRIP-seq analysis services do you offer?
    • Basic Analysis Advanced Analysis
      Sequencing data quality assessment Differential expression analysis of methylated genes
      Quality control Differential methylation gene GO function enrichment analysis
      Gene coverage analysis Differential methylation gene KEGG pathway analysis
      m6A peaks identification Differential methylation gene reactome pathway analysis
      m6A peaks feature annotation Differential expression analysis of methylated lncRNAs
      m6A mRNA analysis GO functional enrichment analysis of differentially methylated lncRNAs cis-encoding genes
      Motif analysis of methylated mRNAs KEGG pathway analysis of cis-coding genes of differentially methylated lncRNAs
      m6A lncRNA analysis Reactome pathway analysis of cis-encoding genes of differentially methylated lncRNAs
      Motif analysis of methylated lncRNA Differential expression analysis of methylated circRNAs
      circRNA recognition and identification analysis GO functional enrichment analysis of differentially methylated circRNA host genes
      m6A mRNA enrichment analysis of circRNA KEGG pathway analysis of differentially methylated circRNA host genes
      Motif analysis of methylated circRNA Reactome pathway analysis of differentially methylated circRNA host genes
  • What are the subsequent experimental validations of MeRIP-seq?
    • MeRIP-qPCR. After enriching RNA with methylation modifications using m6A antibody, the next step is to quantify the enriched RNA directly using qPCR. It is recommended to combine with gene expression RT-qPCR, WB and other validation means together to verify the test results.
  • Is it necessary to do MeRIP-seq in addition to normal transcriptome sequencing?
  • Why is it necessary to do both Input and IP samples for MeRIP-seq?
    • In the MeRIP-seq experimental setup, Input and IP form a pair of samples. The IP samples are specifically enriched with m6A antibody for methylation-modified RNA fragments, while Input is just fragmented RNA as a control to abate background noise, and library building and sequencing are carried out in parallel. Combined peak detection analysis requires integrating data from both samples and using Input data to exclude peaks with high background expression levels or non-specific binding to improve the accuracy of calling peaks.
  • Is there any species restriction to perform MeRIP-seq?
    • If not human, mouse and rat species are recommended to be consulted first. Generally, species with reference genome, genome splicing to chromosome level and complete gtf annotation files can be carried out; eukaryotic, prokaryotic or viral projects are recommended to evaluate first as there are differences in software and parameters involved in binding peak detection.

  • Sample Preparation

  • What are the sample delivery requirements for MeRIP-seq?
    • • Sample type: cell, tissue, total RNA
      • Conventional MeRIP-seq: total RNA ≥ 25 μg after Dnase digestion, concentration ≥ 100 ng/μg, RIN ≥ 8.
      • Other species: Total RNA ≥ 150 μg, concentration ≥ 100 ng/μg, RIN ≥ 8
  • Is it better to use RNA directly or cDNA for MeRIP-seq?
    • After total RNA is extracted put it into NF water and store it at -80°C for at least 2-4 weeks. If mRNA enrichment is done for total RNA, please carry out the experiment immediately within 2 days, otherwise the mRNA will be severely degraded.
      As for cDNA, if there is a long-distance shipping situation exists, it is not recommended to send RNA, or send cDNA mainly. At this time, you can reverse transcribe the product from Input and IP down and put it into NF water for dry ice transport.
For Research Use Only. Not for use in diagnostic procedures.
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MeRIP Sequencing (m6A Analysis)
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