Project description:Define and compare H3K4me2 enrichment in OCI-Ly1 and OCI-Ly7 cell lines. Using ChIP-seq, we examined the H3K4me2 genomic enrichment locations in two biological replicates in each cell line
Project description:Targeted genomic enrichment followed by next-generation sequencing dramatically increased the efficiency of mutation discovery in human genomes. Here we demonstrate that these techniques also revolutionize traditional genetic approaches in model systems. We developed a two-step protocol utilizing a traditional bulk-segregant analysis (BSA) approach for positional cloning mutants in phenotype-driven forward genetic screens. First, BSA pools are 'light' sequenced for rough mapping, followed by targeted enrichment and deep-sequencing of the mutant BSA pool for the linked genomic region to fine-map and discover candidate mutations. We applied this method successfully to three Arabidopsis mutants and show that it can be scaled by multiplexing. Similarly, we applied these techniques to a gene-driven reverse genetics method (chemical driven target-selected mutagenesis or TILLING) that is used for generating gene knockouts in a wide range of organisms, including plants, invertebrates and vertebrates. We developed an efficient multiplexed genomic enrichment protocol for pre-barcoded samples. As a proof-of-principle, 770 genes were screened for induced mutations in 30 rats, which identified all but one known variants (30) as well as a large series of novel knockout and missense alleles. Mutations were retrieved at the expected frequency with a the false-positive rate of less than 1 in 6 million basepairs, which is much lower as compared to traditional mutation discovery approaches. Both methods are largely independent of the genome size due to the targeted enrichment and can thus be applied to any genetic model system of interest. Targeted genomic enrichment followed by next-generation sequencing dramatically increased the efficiency of mutation discovery in human genomes. Here we demonstrate that these techniques also revolutionize traditional genetic approaches in model systems. We developed a two-step protocol utilizing a traditional bulk-segregant analysis (BSA) approach for positional cloning mutants in phenotype-driven forward genetic screens. First, BSA pools are 'light' sequenced for rough mapping, followed by targeted enrichment and deep-sequencing of the mutant BSA pool for the linked genomic region to fine-map and discover candidate mutations. We applied this method successfully to three Arabidopsis mutants and show that it can be scaled by multiplexing. Similarly, we applied these techniques to a gene-driven reverse genetics method (chemical driven target-selected mutagenesis or TILLING) that is used for generating gene knockouts in a wide range of organisms, including plants, invertebrates and vertebrates. We developed an efficient multiplexed genomic enrichment protocol for pre-barcoded samples. As a proof-of-principle, 770 genes were screened for induced mutations in 30 rats, which identified all but one known variants (30) as well as a large series of novel knockout and missense alleles. Mutations were retrieved at the expected frequency with a the false-positive rate of less than 1 in 6 million basepairs, which is much lower as compared to traditional mutation discovery approaches. Both methods are largely independent of the genome size due to the targeted enrichment and can thus be applied to any genetic model system of interest.
Project description:To examine the influences for 5hmC enrichment on genome wide after Tet3 inactivation, we determined the enrichment profiles of 5hmC, the direct demethylated target of Tet3, by hMeDIP-seq with genomic DNA from control and Tet3 inactivated lung smooth muscle cells
Project description:Chromatin Immunoprecipitation followed by high throughput sequencing to identify the location of Orc1, Orc2 and Orc4 genomic enrichment.
Project description:These experiments were designed to test the hypothesis that REST and Polycomb Repressor Complex 2 function cooperatively in undifferentiated ESCs. Our results showed that a majority of REST-bound genomic regions were not associated with H3K27me3 enrichment and loss of H3K27me3 enrichment was not a general observation in REST -/- ESCs. These results support the conclusion that REST and PRC2 function independently in ESCs and similarly contribute to maintaing a transcriptionally poised state through antagonism of H3K4me3. Examination of REST-bound regions in undifferentiated mouse embryonic stem cells (ESC), and a comparison of H3K27me3 distribution between WT and REST-/- ESCs.
Project description:Covalent modification of DNA distinguishes cellular identities and is crucial for regulating the pluripotency and differentiation of embryonic stem (ES) cells. The recent demonstration that 5-methylcytosine (5-mC) may be further modified to 5-hydroxymethylcytosine (5-hmC) in ES cells has revealed a novel regulatory paradigm to modulate the epigenetic landscape of pluripotency. To understand the role of 5-hmC in the epigenomic landscape of pluripotent cells, here we profile the genome-wide 5-hmC distribution and correlate it with the genomic profiles of 11 diverse histone modifications and six transcription factors in human ES cells. By integrating genomic 5-hmC signals with maps of histone enrichment, we link particular pluripotency-associated chromatin contexts with 5-hmC. Intriguingly, through additional correlations with defined chromatin signatures at promoter and enhancer subtypes, we show distinct enrichment of 5-hmC at enhancers marked with H3K4me1 and H3K27ac. These results suggest potential role(s) for 5-hmC in the regulation of specific promoters and enhancers. In addition, our results provide a detailed epigenomic map of 5-hmC from which to pursue future functional studies on the diverse regulatory roles associated with 5-hmC. Genome wide enrichment profile of 5-hmC in H1 human embryonic stem cells
Project description:We report that DNA2 predominantly bound to the centromeric α-satellite regions. The centromeric regions contained 58% of the DNA2-associated DNA, representing a 33.5-fold enrichment over genomic input. To define the under-replicated DNA regions in DNA2-null cells, we conducted whole-genome DNA sequencing of the under-replicated BrdU negative DNA. After normalization to genomic input DNA from the same cells, 12.9% of the peaks from the under-replicated DNA aligned with the centromeric DNA regions, representing an 8.5-fold enrichment. In addition, among the peaks that overlapped between the DNA2 pull-down and under-replicated regions, two thirds fell into the centromeric regions, representing a 48-fold enrichment.