Dataset Information

Genome-wide profiling of nucleosome position and chromatin accessibility in single cells using scMNase-seq.

ABSTRACT: Nucleosome organization is important for chromatin compaction and accessibility. Profiling nucleosome positioning genome-wide in single cells provides critical information to understand the cell-to-cell heterogeneity of chromatin states within a cell population. This protocol describes single-cell micrococcal nuclease sequencing (scMNase-seq), a method for detecting genome-wide nucleosome positioning and chromatin accessibility simultaneously from a small number of cells or single cells. To generate scMNase-seq libraries, single cells are isolated by FACS sorting, lysed and digested by MNase. DNA is purified, end-repaired and ligated to Y-shaped adaptors. Following PCR amplification with indexing primers, the subnucleosome-sized (fragments with a length of ≤80 bp) and mononucleosome-sized (fragments with a length between 140 and 180 bp) DNA fragments are recovered and sequenced on Illumina HiSeq platforms. On average, 0.5-1 million unique mapped reads are obtained for each single cell. The mononucleosome-sized DNA fragments precisely define genome-wide nucleosome positions in single cells, while the subnucleosome-sized DNA fragments provide information on chromatin accessibility. Library preparation of scMNase-seq takes only 2 d, requires only standard molecular biology techniques and does not require sophisticated laboratory equipment. Processing of high-throughput sequencing data requires basic bioinformatics skills and uses publicly available bioinformatics software.

SUBMITTER: Gao W

PROVIDER: S-EPMC10895462 | biostudies-literature | 2020 Jan

REPOSITORIES: biostudies-literature

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Publications

Genome-wide profiling of nucleosome position and chromatin accessibility in single cells using scMNase-seq.

Gao Weiwu W Lai Binbin B Ni Bing B Zhao Keji K

Nature protocols 20191213 1

Nucleosome organization is important for chromatin compaction and accessibility. Profiling nucleosome positioning genome-wide in single cells provides critical information to understand the cell-to-cell heterogeneity of chromatin states within a cell population. This protocol describes single-cell micrococcal nuclease sequencing (scMNase-seq), a method for detecting genome-wide nucleosome positioning and chromatin accessibility simultaneously from a small number of cells or single cells. To gene ...[more]

PMID: 31836865

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Project description:Differential accessibility of DNA to nuclear proteins underlies the regulation of numerous cellular processes. Although DNA accessibility is primarily determined by the presence or absence of nucleosomes, differences in nucleosome composition or dynamics may also regulate accessibility. Methods for mapping nucleosome positions and occupancies genome-wide (MNase-seq) have uncovered the nucleosome landscapes of many different cell types and organisms. Conversely, methods specialized for the detection of large nucleosome-free regions of chromatin (DNase-seq, FAIRE-seq) have uncovered numerous gene regulatory elements. However, these methods are less successful in measuring the accessibility of DNA sequences within nucelosome arrays.Here we probe the genome-wide accessibility of multiple cell types in an unbiased manner using restriction endonuclease digestion of chromatin coupled to deep sequencing (RED-seq). Using this method, we identified differences in chromatin accessibility between populations of cells, not only in nucleosome-depleted regions of the genome (e.g., enhancers and promoters), but also within the majority of the genome that is packaged into nucleosome arrays. Furthermore, we identified both large differences in chromatin accessibility in distinct cell lineages and subtle but significant changes during differentiation of mouse embryonic stem cells (ESCs). Most significantly, using RED-seq, we identified differences in accessibility among nucleosomes harboring well-studied histone variants, and show that these differences depend on factors required for their deposition.Using an unbiased method to probe chromatin accessibility genome-wide, we uncover unique features of chromatin structure that are not observed using more widely-utilized methods. We demonstrate that different types of nucleosomes within mammalian cells exhibit different degrees of accessibility. These findings provide significant insight into the regulation of DNA accessibility.

Dataset Information

Genome-wide profiling of nucleosome position and chromatin accessibility in single cells using scMNase-seq.

Publications

Genome-wide profiling of nucleosome position and chromatin accessibility in single cells using scMNase-seq.

Similar Datasets

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