Simultaneous Profiling of mRNA Transcriptome and DNA Methylome from a Single Cell
Single-cell transcriptome and single-cell methylome analysis have successfully revealed the heterogeneity in transcriptome and DNA methylome between single cells, and have become powerful tools to understand the dynamics of transcriptome and DNA methylome
- PDF / 240,578 Bytes
- 15 Pages / 504.567 x 720 pts Page_size
- 42 Downloads / 172 Views
duction DNA methylation is among the best studied epigenetic modification, which has been shown to be related to many critical biological progress [1–3]. Integrative analysis of bisulfite sequencing data
Youjin Hu, Qin An, and Ying Guo contributed equally to this work. Valentina Proserpio (ed.), Single Cell Methods: Sequencing and Proteomics, Methods in Molecular Biology, vol. 1979, https://doi.org/10.1007/978-1-4939-9240-9_21, © Springer Science+Business Media, LLC, part of Springer Nature 2019
363
364
Youjin Hu et al.
with RNA-seq data revealed how DNA methylation correlates with genes’ expression level and a measurement of the relationship between DNA methylation and RNA transcription with higher resolution will inspire a more faithful hypothesis about how DNA methylation and other epigenetic modification interact with gene expression. Recently, advance in technology has made it possible to profile transcriptome or DNA methylome at single-cell level [4–6], such as single-cell RNA-seq [7–9], single-cell bisulfite sequencing scBS-seq [4], and single-cell reduced representative bisulfite sequencing (scRRBS) [5]. Based on these techniques, growing evidence supporting the existence of transcriptional and epigenetic heterogeneity in previously thought “pure” cell populations, and the heterogeneity in transcriptome and DNA methylome impose a huge challenge to understand the true relationship between them. Recently, we and other groups have developed methods to simultaneously profile DNA methylome and transcriptome from one single cell (scMT-seq) [10–14]. Integrative analysis of these multi-omics from the same single cell can provide detailed subgrouping picture of a complex cell population and accurate temporal cellular roadmap of differentiation progress. scMT-seq includes four steps: (1) the cell membrane is selectively lysed and the nucleus are physically separatedform the cytoplasm; (2) the cytoplasm containing the majority of mRNA is used for single-cell RNA sequencing by Smart-seq2; (3) the nucleus which contains all genomic DNA is used for DNA methylome profiling by single-cell RRBS (scRRBS); (4) next-generation sequencing and data analysis. The experimental part of the protocol takes 3 weeks.
2
Materials
2.1 Equipment and Consumables
1. High Sensitivity DNA Kit Chips and Reagents (Agilent Technologies). 2. Qubit dsDNA HS Assay Kit (Thermo Fisher). 3. MethyCode kit (Thermo Scientific). Other bisulfite conversion kits can also be used.
2.2 Solutions (The Solutions Were Prepared for Ten Reactions)
1. Cell membrane-selective lysis buffer: 2% Triton-X100 19 μl, 40 U/μl RNase inhibitor 1 μl, 10 μM oligo-dT30VN primer 10 μl, and 10 mM dNTP 10 μl. Pipet 4 μl lysis buffer to each PCR tube for one sample. 2. RRBS lysis buffer: Protease 11.25 μl and 1.2 pg/μl lambda DNA (dam–, dcm–; Thermo Scientific, cat. no. SD0021) 0.55 μl.
mRNA Transcriptome and DNA Methylome from a Single Cell
365
3. RT mix: SuperScript III first-strand buffer 20 μl, SuperScript III reverse transcriptase (ThermoFisher, 18080044) 5 μl, 4
Data Loading...
