Project description: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 recently that will 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. Last year we reported a new method for profiling the DNA methylome and transcriptome from the same single cell. Here we present details of the protocol and provide guidance on its utility. Our Smart-RRBS protocol entails physically separating mRNA and genomic DNA and combines Smart-Seq2 and Reduced Representation Bisulfite Sequencing (RRBS). It generates paired epigenetic promoter and RNA-expression measurements for a substantial fraction (approximately one-fourth) of protein-coding genes in a typical single cell. It also works for micro-dissected tissue samples comprising hundreds of cells. The protocol, excluding sequencing, takes ~3 days to process up to 192 samples manually
Project description: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 recently that will 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. Last year we reported a new method for profiling the DNA methylome and transcriptome from the same single cell. Here we present details of the protocol and provide guidance on its utility. Our Smart-RRBS protocol entails physically separating mRNA and genomic DNA and combines Smart-Seq2 and Reduced Representation Bisulfite Sequencing (RRBS). It generates paired epigenetic promoter and RNA-expression measurements for a substantial fraction (approximately one-fourth) of protein-coding genes in a typical single cell. It also works for micro-dissected tissue samples comprising hundreds of cells. The protocol, excluding sequencing, takes ~3 days to process up to 192 samples manually
Project description:Improved Smart-Seq for sensitive full-length transcriptome profiling in single cells. Cells of four different origins were profiled using commercial SMARTer and compared to five variants of an improved protocol (Smart-Seq2).
Project description:We develop a single cell methylome analysis technique based on RRBS, which works robustly for mouse embryonic stem cells (mESCs), sperm, metaphase II oocytes, and zygotes. In total, 36 samples were analyzed, including 8 single mouse embryonic stem cells (mESCs), pooled-5, 10, 20 mESCs, bulk mESCs, 7 single sperms, 10 single pronuclei from 5 individual zygotes, 2 metaphase II oocytes, 2 the first polar bodies and 3 negative controls.
Project description:We develop a single cell methylome analysis technique based on RRBS, which works robustly for mouse embryonic stem cells (mESCs), sperm, metaphase II oocytes, and zygotes.