Smart-RRBS for single-cell methylome and transcriptome analysis.
Amino Acid Sequence
Anti-Bacterial Agents
/ pharmacology
DNA
/ metabolism
DNA (Cytosine-5-)-Methyltransferases
/ genetics
Doxycycline
/ pharmacology
Epigenome
Gene Expression Regulation
/ drug effects
Humans
Intracellular Signaling Peptides and Proteins
RNA, Messenger
/ genetics
Single-Cell Analysis
Transcriptome
Journal
Nature protocols
ISSN: 1750-2799
Titre abrégé: Nat Protoc
Pays: England
ID NLM: 101284307
Informations de publication
Date de publication:
08 2021
08 2021
Historique:
received:
07
09
2020
accepted:
12
05
2021
pubmed:
11
7
2021
medline:
30
9
2021
entrez:
10
7
2021
Statut:
ppublish
Résumé
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.
Identifiants
pubmed: 34244697
doi: 10.1038/s41596-021-00571-9
pii: 10.1038/s41596-021-00571-9
pmc: PMC8672372
mid: NIHMS1760767
doi:
Substances chimiques
Anti-Bacterial Agents
0
FILIP1L protein, human
0
Intracellular Signaling Peptides and Proteins
0
RNA, Messenger
0
DNA
9007-49-2
DNA (Cytosine-5-)-Methyltransferases
EC 2.1.1.37
Doxycycline
N12000U13O
Types de publication
Journal Article
Research Support, N.I.H., Extramural
Research Support, Non-U.S. Gov't
Review
Langues
eng
Sous-ensembles de citation
IM
Pagination
4004-4030Subventions
Organisme : NCI NIH HHS
ID : K99 CA248955
Pays : United States
Organisme : NIGMS NIH HHS
ID : P01 GM099117
Pays : United States
Organisme : NCI NIH HHS
ID : R01 CA229902
Pays : United States
Informations de copyright
© 2021. The Author(s), under exclusive licence to Springer Nature Limited.
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