ABSTRACT: Transcription profiling by high throughput sequencing of mouse embryonic fibroblasts, terminally differentiated neurons and embryonic stem cells
Project description:High throughput sequencing of poly-A RNA from control- and sporadic Parkinson´s disease patient derived fibroblasts (n=15), induced pluripotent stem cells (n=31) and differentiated midbrain neurons (n=15). Fibroblasts and iPSCs do not show major differences on single gene level. In contrast, midbrain neurons derived from Parkinson´s disease patients show changes known to be associated with neurodegenerative diseases.
Project description:Embryonic stem cells (ESCs) and induced-pluripotent stem cells (iPSCs) self-renew and differentiate into an array of cell types in vitro and in vivo. A complex network of genetic and epigenetic pathways regulates the self-renewal and differentiation of these pluripotent cells, and the structure and covalent modifications of chromatin play a prominent role in this process. We examine nucleosome occupancy in mouse and human embryonic stem cells (ESCs), induced-pluripotent stem cells (iPSCs), and differentiated cell types using MNase-seq. To address variability inherent in this technique, we developed a bioinformatic approach that enabled the identification of regions of difference (RoD) in nucleosome occupancy between pluripotent and somatic cells. The majority of changes in nucleosomal signatures that occur in differentiation are reset during reprogramming. We conclude that changes in nucleosome occupancy are a hallmark of pluripotency and likely identify key regulatory regions that play a role in determining cell identity. Micrococcal nuclease digestion of chromatin in crosslinked cells was followed by high throughput sequencing. These experiments were carried out in four mouse cell types: embryonic stem cells, induced pluripotent stem cells, somatic tail-tip fibroblasts and liver, and three human cell types: H1-OGN embryonic stem cells, H1-OGN induced pluripotent stem cells, and fibroblasts differentiated from H1-OGN ESCs. At least two replicates performed with each cell type were sequenced.
Project description:Chromatin immunoprecipitation against CTCF followed by Illumina High-throughput sequencing. Examination of CTCF binding in embryonic stem cells and embryonic fibroblasts
Project description:To understand the mechanism underlying the versatility in transcriptional regulation by Sox2, we compared genome-wide binding sites of Sox2 in embryonic stem (ES) cells and trophoblast stem (TS) cells by chromatin immunoprecipitation followed by high-throughput sequencing (ChIP-seq). A tetracycline-inducible Oct3/4 knockout ES cell line ZHBTc4 was treated with Tet for 4 days in the presence of FGF4 and mouse embryonic fibroblasts (MEFs).
Project description:We identified FMRP-bound RNAs in hPSC (human pluripotent stem cells)-differentiated forebrain neuroprogenitors (NPCs) and neurons using crosslinking immunoprecipitation (CLIP) coupled to high-throughput sequencing. We examined transcriptomic and proteomic changes in FMRP-KO NPCs and neurons.
Project description:To understand the mechanism underlying the transcriptional regulation by Sox2, we analyzed genome-wide binding sites of Sox2, Tfap2c, and Cdx2 in trophoblast stem (TS) cells by chromatin immunoprecipitation followed by high-throughput sequencing (ChIP-seq). ZHBTc4- and embryo-derived TS cell lines were maintained in the presence of FGF4 and mouse embryonic fibroblasts (MEFs).
Project description:High throughput sequencing of small RNAs (mature miRNA and piRNA/piRNA-like molecules) from control- and sporadic Parkinson´s disease patient derived fibroblasts (n=15), induced pluripotent stem cells (n=26) and differentiated midbrain neurons (n=10). In parallel, cingulate gyrus samples from eight control- and eight Parkinson´s disease patients obtained from the Netherlands Brain Bank were sequenced. We found significant differences between the groups in every cell type and in tissue.