Project description:DNA methylation is a complex epigenetic marker that can be analysed using a wide variety of methods. Interpretation and visualisation of DNA methylation data can mask complexity in terms of methylation status at each CpG site, cellular heterogeneity of samples and allelic DNA methylation patterns within a given DNA strand. Bisulfite sequencing is considered the gold standard, however visualisation of massively parallel sequencing results remains a significant challenge. We created a program called Methpat that facilitates visualisation and interpretation of bisulfite sequencing data generated by massively parallel sequencing. To demonstrate this, we performed multiplex PCR that targeted 48 regions of interest across 95 human samples. The regions selected included known gene promoters associated with cancer, repetitive elements, known imprinted regions and mitochondrial genomic sequences. We interrogated a range of samples including human cell lines, primary tumours and primary tissue samples. Methpat generates two forms of output: a tab delimited text file for each sample that summarises DNA methylation patterns and their read counts for each amplicon and a HTML file that summarises this data visually. Methpat can be used with publicly available whole genome bisulfite sequencing (WGBS) and reduced representation bisulfite sequencing (RRBS) datasets with sufficient read depths. Using Methpat, complex DNA methylation data derived from massively parallel sequencing can be summarised and visualised for biological interpretation. By accounting for allelic DNA methylation states and their abundance in a sample, Methpat can unmask the complexity of DNA methylation and reveal further biological insight in existing datasets. Multiplex bisulfite PCR and Next Generation sequencing of primary human samples and breast cancer cell lines.
Project description:Chip-Exo of various factors (BRD4, DNMT1, H3K27ac and Tet3) in ES cells under either serum conditions or 2i conditions. TET3 chip-exo was also performed from mouse E13.5 brain Factors were identified as important epigenetic regulators in serum to 2i transition where cells undergo global demethylation and exhibit a more nave stem cell state..
Project description:This data was generated by ENCODE. If you have questions about the data, contact the submitting laboratory directly (Richard Sandstrom mailto:email@example.com). If you have questions about the Genome Browser track associated with this data, contact ENCODE (mailto:firstname.lastname@example.org). This track is produced as part of the ENCODE Project. This track shows DNaseI sensitivity measured genome-wide in different cell lines using the Digital DNaseI methodology (see below), and DNaseI hypersensitive sites. DNaseI has long been used to map general chromatin accessibility and DNaseI hypersensitivity is a universal feature of active cis-regulatory sequences. The use of this method has led to the discovery of functional regulatory elements that include enhancers, insulators, promotors, locus control regions and novel elements. For each experiment (cell type) this track shows DNaseI sensitivity as a continuous function using sequencing tag density (Raw Signal), and discrete loci of DNaseI sensitive zones (HotSpots) and hypersensitive sites (Peaks)." For data usage terms and conditions, please refer to http://www.genome.gov/27528022 and http://www.genome.gov/Pages/Research/ENCODE/ENCODEDataReleasePolicyFinal2008.pdf Cells were grown according to the approved ENCODE cell culture protocols. Digital DNaseI was performed by DNaseI digestion of intact nuclei, isolating DNaseI 'double-hit' fragments as described in Sabo et al. (2006), and direct sequencing of fragment ends (which correspond to in vivo DNaseI cleavage sites) using the Solexa platform (36 bp reads). Uniquely mapping high-quality reads were mapped to the genome. DNaseI sensitivity is directly reflected in raw tag density (Raw Signal), which is shown in the track as density of tags mapping within a 150 bp sliding window (at a 20 bp step across the genome). DNaseI sensitive zones (HotSpots) were identified using the HotSpot algorithm described in Sabo et al. (2004). 1.0% false discovery rate thresholds (FDR 0.01) were computed for each cell type by applying the HotSpot algorithm to an equivalent number of random uniquely mapping 36mers. DNaseI hypersensitive sites (DHSs or Peaks) were identified as signal peaks within FDR 1.0% hypersensitive zones using a peak-finding algorithm.
Project description:The mammalian CCCTC-binding factor (CTCF) regulates gene expression through the formation of higher order chromatin structures. Recent evidence has implicated a role for CTCF in regulating gene expression in the human MHCII locus. To investigate the role of CTCF in murine MHCII gene expression we mapped CTCF binding sites in B cells (MHCII+ cells) and Plasmablasts which are differentiated B cells that have silenced MHCII gene expression. These observations lead to the identification of differential CTCF binding during differentiation in these cell types and suggest mechanims of MHCII gene regulation. Comparison of CTCF binding in B cells and Plasmablasts in mice using ChIP-seq
Project description:These data include the genome wide occupancy of histone modifications and transcription factors by ChIP sequencing in mouse crypt cells and in mouse ISCs. Immuno-precipitation of formaldehyde cross-linked chromatin prepared from wild type and Ring1a Ring1b dKO crypt cells and from WT ISCs using specific antibody against different target protein/modification.
Project description:We report ChIP-Seq data for GATA1 and the leukemia-associated short isoform GATA1s in G1ME cells, a Gata1-null cell line with both erythroid and megakaryocytic differentiation potential. We introduced HA-tagged GATA1 or GATA1s into G1ME cells via retroviral transduction. The cells were crosslinked at 48h post-transduction, and an HA antibody was used for chromatin immunoprecipitation (ChIP). ChIP and input samples were sequenced on Illumina GAII or GAIIx high-throughput sequencers. The data reveal a deficiency of chromatin occupancy by GATA1s, especially at genes involved in erythrocyte differentiation and function. Examinaton of chromatin occupancy of GATA1 and GATA1s in G1ME cells cultured in TPO.