Refined RIP-seq protocol for epitranscriptome analysis with low input materials.

PLoS Biology
Yong ZengHousheng Hansen He

Abstract

N6-Methyladenosine (m6A) accounts for approximately 0.2% to 0.6% of all adenosine in mammalian mRNA, representing the most abundant internal mRNA modifications. m6A RNA immunoprecipitation followed by high-throughput sequencing (MeRIP-seq) is a powerful technique to map the m6A location transcriptome-wide. However, this method typically requires 300 μg of total RNA, which limits its application to patient tumors. In this study, we present a refined m6A MeRIP-seq protocol and analysis pipeline that can be applied to profile low-input RNA samples from patient tumors. We optimized the key parameters of m6A MeRIP-seq, including the starting amount of RNA, RNA fragmentation, antibody selection, MeRIP washing/elution conditions, methods for RNA library construction, and the bioinformatics analysis pipeline. With the optimized immunoprecipitation (IP) conditions and a postamplification rRNA depletion strategy, we were able to profile the m6A epitranscriptome using 500 ng of total RNA. We identified approximately 12,000 m6A peaks with a high signal-to-noise (S/N) ratio from 2 lung adenocarcinoma (ADC) patient tumors. Through integrative analysis of the transcriptome, m6A epitranscriptome, and proteome data in the same patient tumors, w...Continue Reading

References

Oct 1, 1974·Proceedings of the National Academy of Sciences of the United States of America·R DesrosiersF Rottman
Feb 16, 2006·BioEssays : News and Reviews in Molecular, Cellular and Developmental Biology·David RatelDidier Wion
Sep 19, 2008·Genome Biology·Yong ZhangX Shirley Liu
Jun 10, 2009·Bioinformatics·Heng LiUNKNOWN 1000 Genome Project Data Processing Subgroup
Jul 20, 2010·Bioinformatics·W J KentD Karolchik
May 6, 2011·Bioinformatics·Timothy L Bailey
Mar 14, 2012·Nature Reviews. Genetics·Christine Vogel, Edward M Marcotte
May 12, 2012·Nature·Dan DominissiniGideon Rechavi
Jun 30, 2012·Bioinformatics·Liguo WangWei Li
Sep 8, 2012·Genome Research·Jennifer HarrowTim J Hubbard
Oct 30, 2012·Bioinformatics·Alexander DobinThomas R Gingeras
Apr 17, 2013·Bioinformatics·Jia MengYufei Huang
Feb 1, 2014·Nature Cell Biology·Shuibin Lin, Richard I Gregory
Sep 28, 2014·Bioinformatics·Simon AndersWolfgang Huber
Dec 2, 2014·Cell Reports·David A OrlandoMatthew G Guenther
Dec 18, 2014·Genome Biology·Michael I LoveSimon Anders
Mar 25, 2015·Nature·Claudio R AlarcónSohail F Tavazoie
Jun 13, 2015·Nucleic Acids Research·Xin DengChuan He
Feb 4, 2016·Nature Structural & Molecular Biology·Nian Liu, Tao Pan
Feb 16, 2016·Molecular Cell·Wen XiaoYun-Gui Yang
Mar 24, 2016·Proceedings of the National Academy of Sciences of the United States of America·Chuanzhao ZhangGregg L Semenza
Apr 28, 2016·Molecular Cell·Shuibin LinRichard I Gregory
Nov 4, 2016·Nature Reviews. Molecular Cell Biology·Boxuan Simen ZhaoChuan He
Jan 14, 2017·Genome Medicine·Samie R Jaffrey, Michael G Kharas
Jan 21, 2017·Cell Research·Ang LiYun-Gui Yang
Feb 2, 2017·Nature Reviews. Molecular Cell Biology·Cole J T LewisAuinash Kalsotra
May 14, 2017·Trends in Genetics : TIG·Jean-Yves Roignant, Matthias Soller

❮ Previous
Next ❯

Citations

Jul 10, 2019·Annual Review of Virology·Graham D WilliamsStacy M Horner
Sep 25, 2019·Nature Methods·Kate D Meyer
Apr 27, 2019·Frontiers in Oncology·Zaira IannielloAlessandro Fatica
Jul 31, 2019·Nature Cell Biology·Xin ChenYun-Gui Yang
Jan 9, 2020·Nucleic Acids Research·Sanqi AnJinkai Wang
Aug 28, 2020·Frontiers in Cell and Developmental Biology·Meng ZhangZiyi Li
Apr 22, 2020·Scientific Reports·Alexa B R McIntyreChristopher E Mason
Nov 28, 2020·Nature Communications·Alexander M PriceMatthew D Weitzman
Oct 17, 2020·Toxicological Sciences : an Official Journal of the Society of Toxicology·Neelakanteswar Aluru, Sibel I Karchner
Jan 30, 2021·Cell Research·Jun'e LiuCheng-Feng Qin
Feb 20, 2021·Briefings in Functional Genomics·Veronica Bergo, Eirini Trompouki
Jun 3, 2021·Non-coding RNA·Venkata Naga Srikanth Garikipati, Shizuka Uchida
May 16, 2021·Journal of Molecular and Cellular Cardiology·Walid Khalid SweaadUNKNOWN EU-CardioRNA COST Action CA17129
Jun 8, 2021·Frontiers in Cellular Neuroscience·Justine MathouxGary P Brennan
Sep 3, 2021·Journal of Genetics and Genomics = Yi Chuan Xue Bao·Tao ChenGuan-Zheng Luo
Feb 28, 2021·Hepatology : Official Journal of the American Association for the Study of Liver Diseases·Tianhong SuMing Kuang
Jun 26, 2021·Genes & Development·Hannah M BurgessIan Mohr

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Methods Mentioned

BETA
immunoprecipitation
pull-down
MeRIP-seq
RNA-Seq
reverse transcription PCR
xenograft
PCR
Assay
pulldown
electrophoresis

Software Mentioned

bcl2fastq
GENCODE
SMARTer
MACS
exomePeak
DESeq2
samtools
DREME
STAR
Andromeda

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