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  Smart-RRBS for single-cell methylome and transcriptome analysis

Gu, H., Raman, A. T., Wang, X., Gaiti, F., Chaligne, R., Mohammad, A. W., et al. (2021). Smart-RRBS for single-cell methylome and transcriptome analysis. Nature Protocols, 16, 4004-4030. doi:10.1038/s41596-021-00571-9.

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Gu, Hongcang, Author
Raman, Ayush T. , Author
Wang, Xiaoxue , Author
Gaiti, Federico , Author
Chaligne, Ronan , Author
Mohammad, Arman W. , Author
Arczewska, Aleksandra Alicja1, Author              
Smith, Zachary D. , Author
Landau, Dan A. , Author
Aryee, Martin J., Author
Meissner, Alexander1, 2, 3, Author              
Gnirke, Andreas, Author
Affiliations:
1Dept. of Genome Regulation (Head: Alexander Meissner), Max Planck Institute for Molecular Genetics, Max Planck Society, ou_2379694              
2Broad Institute of MIT and Harvard, Cambridge, MA, USA, ou_persistent22              
3Department of Stem Cell and Regenerative Biology, Harvard University, Cambridge, MA, USA, ou_persistent22              

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 Abstract: The integration of DNA methylation and transcriptional state within single cells is of broad interest. Several single-cell dual- and multi-omics approaches have been reported that enable further investigation into cellular heterogeneity, including the discovery and in-depth study of rare cell populations. Such analyses will continue to provide important mechanistic insights into the regulatory consequences of epigenetic modifications. We recently reported a new method for profiling the DNA methylome and transcriptome from the same single cells in a cancer research study. Here, we present details of the protocol and provide guidance on its utility. Our Smart-RRBS (reduced representation bisulfite sequencing) protocol combines Smart-seq2 and RRBS and entails physically separating mRNA from the genomic DNA. It generates paired epigenetic promoter and RNA-expression measurements for ~24% of protein-coding genes in a typical single cell. It also works for micro-dissected tissue samples comprising hundreds of cells. The protocol, excluding flow sorting of cells and sequencing, takes ~3 d to process up to 192 samples manually. It requires basic molecular biology expertise and laboratory equipment, including a PCR workstation with UV sterilization, a DNA fluorometer and a microfluidic electrophoresis system.

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Language(s): eng - English
 Dates: 2021-05-122021-07-092021-09
 Publication Status: Published in print
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 Rev. Type: -
 Identifiers: DOI: 10.1038/s41596-021-00571-9
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Title: Nature Protocols
  Other : Nat. Protoc.
Source Genre: Journal
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Publ. Info: London, UK : Nature Publishing Group
Pages: 17 Volume / Issue: 16 Sequence Number: - Start / End Page: 4004 - 4030 Identifier: ISSN: 1750-2799
CoNE: https://pure.mpg.de/cone/journals/resource/1000000000223800_1