Comparative analysis of improved m6A sequencing based on antibody optimization for low-input samples
Abstract The most effective method for mapping N6-methyladenosine (m6A) is m6A RNA immunoprecipitation sequencing (MeRIP-seq). The quality of MeRIP-seq relies on various factors, with the anti-m6A antibody being a crucial determinant. However, comprehensive research on anti-m6A antibody selection an...
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Nature Portfolio
2025-01-01
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| Series: | Scientific Reports |
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| Online Access: | https://doi.org/10.1038/s41598-025-85150-8 |
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| author | Jiafeng Lu Wenjuan Xia Jincheng Li Liya Zhang Chunfeng Qian Hong Li Boxian Huang |
| author_facet | Jiafeng Lu Wenjuan Xia Jincheng Li Liya Zhang Chunfeng Qian Hong Li Boxian Huang |
| author_sort | Jiafeng Lu |
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| description | Abstract The most effective method for mapping N6-methyladenosine (m6A) is m6A RNA immunoprecipitation sequencing (MeRIP-seq). The quality of MeRIP-seq relies on various factors, with the anti-m6A antibody being a crucial determinant. However, comprehensive research on anti-m6A antibody selection and optimal concentrations for different tissues has been limited. In this study, we optimized the concentration of five different anti-m6A antibodies across various tissues. Our findings demonstrated that 5 µg of Millipore antibodies (ABE572 and MABE1006) performed well, starting from 15 µg total RNA from the liver, while 1.25 µg of Cell Signaling Technology antibodies (CST) (#56593) was suitable for low-input total RNA. In summary, we provide a significant guideline for anti-m6A antibody selection in MeRIP sequencing for different tissues, especially in the context of low-input RNA. |
| format | Article |
| id | doaj-art-08a5ca11f54a43878007c9337d3675f5 |
| institution | Kabale University |
| issn | 2045-2322 |
| language | English |
| publishDate | 2025-01-01 |
| publisher | Nature Portfolio |
| record_format | Article |
| series | Scientific Reports |
| spelling | doaj-art-08a5ca11f54a43878007c9337d3675f52025-01-12T12:23:06ZengNature PortfolioScientific Reports2045-23222025-01-0115111210.1038/s41598-025-85150-8Comparative analysis of improved m6A sequencing based on antibody optimization for low-input samplesJiafeng Lu0Wenjuan Xia1Jincheng Li2Liya Zhang3Chunfeng Qian4Hong Li5Boxian Huang6State Key Laboratory of Reproductive Medicine, Suzhou Municipal Hospital, Suzhou Affiliated Hospital of Nanjing Medical University, Gusu School, Nanjing Medical UniversityState Key Laboratory of Reproductive Medicine, Suzhou Municipal Hospital, Suzhou Affiliated Hospital of Nanjing Medical University, Gusu School, Nanjing Medical UniversityState Key Laboratory of Reproductive Medicine, Suzhou Municipal Hospital, Suzhou Affiliated Hospital of Nanjing Medical University, Gusu School, Nanjing Medical UniversityState Key Laboratory of Reproductive Medicine, Suzhou Municipal Hospital, Suzhou Affiliated Hospital of Nanjing Medical University, Gusu School, Nanjing Medical UniversityState Key Laboratory of Reproductive Medicine, Suzhou Municipal Hospital, Suzhou Affiliated Hospital of Nanjing Medical University, Gusu School, Nanjing Medical UniversityState Key Laboratory of Reproductive Medicine, Suzhou Municipal Hospital, Suzhou Affiliated Hospital of Nanjing Medical University, Gusu School, Nanjing Medical UniversityState Key Laboratory of Reproductive Medicine, Suzhou Municipal Hospital, Suzhou Affiliated Hospital of Nanjing Medical University, Gusu School, Nanjing Medical UniversityAbstract The most effective method for mapping N6-methyladenosine (m6A) is m6A RNA immunoprecipitation sequencing (MeRIP-seq). The quality of MeRIP-seq relies on various factors, with the anti-m6A antibody being a crucial determinant. However, comprehensive research on anti-m6A antibody selection and optimal concentrations for different tissues has been limited. In this study, we optimized the concentration of five different anti-m6A antibodies across various tissues. Our findings demonstrated that 5 µg of Millipore antibodies (ABE572 and MABE1006) performed well, starting from 15 µg total RNA from the liver, while 1.25 µg of Cell Signaling Technology antibodies (CST) (#56593) was suitable for low-input total RNA. In summary, we provide a significant guideline for anti-m6A antibody selection in MeRIP sequencing for different tissues, especially in the context of low-input RNA.https://doi.org/10.1038/s41598-025-85150-8m6A antibodyMeRIP SeqHuman fetal brainHuman fetal liverMouse liverMouse brain |
| spellingShingle | Jiafeng Lu Wenjuan Xia Jincheng Li Liya Zhang Chunfeng Qian Hong Li Boxian Huang Comparative analysis of improved m6A sequencing based on antibody optimization for low-input samples Scientific Reports m6A antibody MeRIP Seq Human fetal brain Human fetal liver Mouse liver Mouse brain |
| title | Comparative analysis of improved m6A sequencing based on antibody optimization for low-input samples |
| title_full | Comparative analysis of improved m6A sequencing based on antibody optimization for low-input samples |
| title_fullStr | Comparative analysis of improved m6A sequencing based on antibody optimization for low-input samples |
| title_full_unstemmed | Comparative analysis of improved m6A sequencing based on antibody optimization for low-input samples |
| title_short | Comparative analysis of improved m6A sequencing based on antibody optimization for low-input samples |
| title_sort | comparative analysis of improved m6a sequencing based on antibody optimization for low input samples |
| topic | m6A antibody MeRIP Seq Human fetal brain Human fetal liver Mouse liver Mouse brain |
| url | https://doi.org/10.1038/s41598-025-85150-8 |
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