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Single-Cell Whole-Genome Bisulfite Sequencing for Analysis of Epigenetic Heterogeneity

ABSTRACT: We report a single-cell whole-genome bisulfite sequencing method (scBS-Seq) capable of accurately measuring DNA methylation at up to 36% of CpGs. We observed that ESCs grown in serum/LIF or 2i/LIF both display epigenetic heterogeneity, with “2i-like” cells present in serum cultures. In silico integration of 12 individual MII oocytes datasets recapitulates the whole DNA methylome, making scBS-Seq a versatile tool to explore DNA methylation in rare cells and heterogeneous populations.

ORGANISM(S): Mus musculus

PROVIDER: GSE56879 | GEO | 2014/07/18

SECONDARY ACCESSION(S): PRJNA244869

REPOSITORIES: GEO

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Single-Cell Whole-Genome Bisulfite Sequencing for Analysis of Epigenetic Heterogeneity

Project description:We report a single-cell whole-genome bisulfite sequencing method (scBS-Seq) capable of accurately measuring DNA methylation at up to 36% of CpGs. We observed that ESCs grown in serum/LIF or 2i/LIF both display epigenetic heterogeneity, with M-bM-^@M-^\2i-likeM-bM-^@M-^] cells present in serum cultures. In silico integration of 12 individual MII oocytes datasetsM-BM- recapitulates the whole DNA methylome, making scBS-Seq a versatile tool to explore DNA methylation in rare cells and heterogeneous populations. scBS-Seq has been performed on mouse MII oocytes for technical validation and on mouse ESCs cultured in 2i/LIF and serum/LIF to investage epigenetic heterogeneity

2014-07-18 | E-GEOD-56879 | biostudies-arrayexpress

Whole-genome bisulfite sequencing of two distinct interconvertible DNA methylomes of mouse embryonic stem cells

2013-07-12 | GSE41923 | GEO

Distinct DNA methylomes of 2i and serum mouse ES cells are interconvertible and reflect pre- and post-implantation embryonic states

Project description:This study describes the DNA methylation profiling using whole-genome bisulfite sequencing of mouse ES cells, either derived and maintained in 2i serum-free NDiff medium, or in the presence of serum and LIF, or maintained and derived in the presence of serum and LIF and subsequently adapted to 2i serum-free NDiff medium, or maintained and derived in the presence of 2i and LIF and subsequently adapted to 2i serum. DNA methylation profiling using whole-genome bisulfite sequencing of 14 samples, 3 different lines (E14, XT67E1, Rex/GFP-2i) of pluripotent mouse ES cells as well during conversion from 2i to serum and vice versa.

2013-07-12 | E-GEOD-41923 | biostudies-arrayexpress

Integrative Proteomic Profiling Reveals PRC2-Dependent Epigenetic Crosstalk Maintains Ground-State Pluripotency.

Project description:The pluripotent ground state is defined as a basal state free of epigenetic restrictions, which influence lineage specification. While naive embryonic stem cells (ESCs) can be maintained in a hypomethylated state with open chromatin when grown using two small-molecule inhibitors (2i)/leukemia inhibitory factor (LIF), in contrast to serum/LIF-grown ESCs that resemble early post-implantation embryos, broader features of the ground-state pluripotent epigenome are not well understood. We identified epigenetic features of mouse ESCs cultured using 2i/LIF or serum/LIF by proteomic profiling of chromatin-associated complexes and histone modifications. Polycomb-repressive complex 2 (PRC2) and its product H3K27me3 are highly abundant in 2i/LIF ESCs, and H3K27me3 is distributed genome-wide in a CpG-dependent fashion. Consistently, PRC2-deficient ESCs showed increased DNA methylation at sites normally occupied by H3K27me3 and increased H4 acetylation. Inhibiting DNA methylation in PRC2-deficient ESCs did not affect their viability or transcriptome. Our findings suggest a unique H3K27me3 configuration protects naive ESCs from lineage priming, and they reveal widespread epigenetic crosstalk in ground-state pluripotency.

2018-11-24 | GSE101675 | GEO

Parallel single-cell bisulfite- and RNA-sequencing link transcriptional and epigenetic heterogeneity [Bisulfite-Seq]

Project description:We report scM&T-seq, a method for parallel single-cell genome-wide methylome and transcriptome sequencing, allowing discovery of associations between transcriptional and epigenetic variation. Profiling of 61 mouse embryonic stem cells confirmed known links between DNA methylation and transcription. Notably, the method reveals novel associations between heterogeneous methylation of distal regulatory elements and transcriptional heterogeneity of key pluripotency genes. E14 ES cells were grown in either serum/LIF or 2i culture conditions and separated into single cells. RNA-Seq or Bisulfite-Seq libraries were prepared. This Series includes only the Bisulfite-Seq data. The list of 61 samples that passed QC in both BS-seq and RNA-seq is included in "Supplementary Table 1" of the associated manuscript.

2015-12-24 | E-GEOD-68642 | biostudies-arrayexpress

RNA-seq of triple knockout (TKO) mouse embryonic stem cells: Dnmt1-/-, Dnmt3a-/-, Dnmt3b-/-.

Project description:TKO mouse embryonic stem cells were cultured in serum/LIF and 2i/LIF in order to investigate the role of methylation in the transcriptional regulation of ground state cells (Clone: TKO 121, Tsumura et.al. 2006).

2020-06-19 | GSE130686 | GEO

RNA-seq analysis of ES cell populations with different functional potentials in serum/LIF and 2i/LIF, as well as analysis of the transcriptional effect of LIF in ground state (2i) culture conditions.

Project description:We report that ES cells cultured in ground state (2i and 2i/LIF) culture conditions are heterogeneous and show heterogeneus expression of extraembryonic markers. Using a highly sensitive reporter for the endoderm marker Hex we can sort Hex high and low populations from either serum/LIF or 2i/LIF and demonstrate that they have different functional properties. Here we explored the transcriptional basis of these functional differences and noted that Hex low (HV-) and Hex high (HV+) populations showed more distinct expression profiles in 2i/LIF than in serum/LIF. Additionally in 2i/LIF the HV+ population showed an upregulation of extraembryonic markers (such as trophoblast stem cell specific genes) and also imprinted genes compared to the HV- population, which is not observed when these populations are sorted from serum/LIF. We also analysed the transcriptional effect of LIF in 2i by analysing unsorted ES cells cultured in either 2i alone or 2i with LIF. We observed that the addition of LIF led to an upregulation of extraembryonic markers but did not effect the expression of pluripotency genes, other than Klf4. Additionally, the most significantly upregulated genes from 2i/LIF cultured ES cells compared to 2i cultured ES cells showed the greatest correlation to placental tissue when compared to the GNF tissue specific expression database. This analysis, alongside functional experiments, suggested that HV+ ES cells in 2i/LIF corresponded to an extraembryonically primed population of cells and that the addition of LIF supported this population.

2013-06-11 | GSE45182 | GEO

Parallel single-cell bisulfite- and RNA-sequencing link transcriptional and epigenetic heterogeneity [RNA-seq]

Project description:We report scM&T-seq, a method for parallel single-cell genome-wide methylome and transcriptome sequencing, allowing discovery of associations between transcriptional and epigenetic variation. Profiling of 61 mouse embryonic stem cells confirmed known links between DNA methylation and transcription. Notably, the method reveals novel associations between heterogeneous methylation of distal regulatory elements and transcriptional heterogeneity of key pluripotency genes. E14 ES cells were grown in either serum/LIF or 2i culture conditions and separated into single cells. RNA-Seq or Bisulfite-Seq libraries were prepared. This Series includes only the RNA-Seq data. The list of 61 samples that passed QC in both BS-seq and RNA-seq is included in "Supplementary Table 1" of the associated manuscript.

2015-12-24 | E-GEOD-74534 | biostudies-arrayexpress

RNA-seq analysis of ES cell populations with different functional potentials in serum/LIF and 2i/LIF, as well as analysis of the transcriptional effect of LIF in ground state (2i) culture conditions.

2013-06-11 | E-GEOD-45182 | biostudies-arrayexpress

MEK and GSK3β inhibition drives ES cells into a naive pluripotent state irrespective of their global methylation status

Project description:Mouse embryonic stem cells (mESCs) can convert to a ground state by dual inhibition of MEK and GSK3β signalling in defined media in the presence of LIF (2i). The cellular transition in 2i leads to uniform expression of pluripotency markers and global DNA hypomethylation. Whether hypomethylation is required for achieving ground state or is an outcome of the conversion process is still not clear. Here we show that J1 wild type and hypomethylated mESCs (dnmt3a-/-, dnmt3b-/-, dnmt1-/- [TKO]) lacking DNA methyltransferases undergo similar morphological and expression changes upon culturing in 2i that are consistent with conversion of both to a more naive state. Maintenance of global DNA methylation levels in 2i media by constitutive expression of de novo methyltransferases is also not a barrier to conversion. Hence, signalling pathways in mES cells regulate pluripotency networks and cell state independently of their global DNA methylation status. Indicated mESCs were cultured in 2i/LIF for 2 weeks and then harvested along with serum/LIF counterparts for microarray analysis. 1ug of total RNA was used to prepare Cy3 labelled aRNA using an Amino Allyl MessageAmp II aRNA kit (Ambion) following manufacturers protocol. The samples were hybridised to SurePrint G3 mouse GE 8x60k microarrays (Agilent) and were scanned using NimbleGen MS 200 (Roche). Results were analysed with custom-written scripts implemented in the statistical programming language R (http://www.R-project.org).

2020-06-16 | GSE72302 | GEO

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