A simple method for generating high-resolution maps of genome wide protein binding
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PROVIDER: PRJNA279984 | ENA |
REPOSITORIES: ENA
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Project description:Chromatin immunoprecipitation (ChIP) and its derivatives are the main techniques used to determine transcription factor binding sites. However, conventional ChIP with sequencing (ChIP-seq) has problems with poor resolution and newer techniques require significant experimental alterations and complex bioinformatics. Here we build upon our high-resolution crosslinking ChIP-seq (X-ChIP-seq) method and compare it to existing methodologies. By using micrococcal nuclease, which has both endo- and exo-nuclease activity to fragment the chromatin and thereby generate precise protein-DNA footprints, high-resolution X-ChIP-seq achieves single base pair resolution of transcription factor binding. A significant advantage of this protocol is the minimal alteration to the conventional ChIP-seq workflow and simple bioinformatic processing. Using High-resolution X-ChIP-seq we determined the genome-wide binding profile of various DNA binding proteins.
2015-06-12 | E-GEOD-67454 | biostudies-arrayexpress
Project description:Chromatin immunoprecipitation (ChIP) and its derivatives are the main techniques used to determine transcription factor binding sites. However, conventional ChIP with sequencing (ChIP-seq) has problems with poor resolution and newer techniques require significant experimental alterations and complex bioinformatics. Here we build upon our high-resolution crosslinking ChIP-seq (X-ChIP-seq) method and compare it to existing methodologies. By using micrococcal nuclease, which has both endo- and exo-nuclease activity to fragment the chromatin and thereby generate precise protein-DNA footprints, high-resolution X-ChIP-seq achieves single base pair resolution of transcription factor binding. A significant advantage of this protocol is the minimal alteration to the conventional ChIP-seq workflow and simple bioinformatic processing.
2015-06-12 | GSE67454 | GEO
Project description:Chromatin immunoprecipitation (ChIP) and its derivatives are the main techniques used to determine transcription factor binding sites. However, conventional ChIP with sequencing (ChIP-seq) has problems with poor resolution, and newer techniques require significant experimental alterations and complex bioinformatics. Previously, we have used a new crosslinking ChIP-seq protocol (X-ChIP-seq) to perform high-resolution mapping of RNA Polymerase II (Skene et al., 2014). Here, we build upon this work and compare X-ChIP-seq to existing methodologies. By using micrococcal nuclease, which has both endo- and exo-nuclease activity, to fragment the chromatin and thereby generate precise protein-DNA footprints, high-resolution X-ChIP-seq achieves single base-pair resolution of transcription factor binding. A significant advantage of this protocol is the minimal alteration to the conventional ChIP-seq workflow and simple bioinformatic processing.
| S-EPMC4480131 | biostudies-literature
Project description:We developed and validated a simple method for viable cryopreservation of whole blood, without any preprocessing, Simple prEservatioN of Single cElls (SENSE), with granulocyte depletion for generating high-quality single-cell profiles. We performed rigorous in-depth characterization of the SENSE method on clinical blood samples and compared it to the conventional multistep density-gradient isolation of peripheral blood mononuclear cells (PBMCs) method. The SENSE method is an effective and simple solution for the cryopreservation of blood samples in clinics/labs and single cell profiles generation.
2023-11-28 | GSE226557 | GEO
Project description:Genome wide patterns of histone modifications in fission yeast Keywords: High resolution maps of nucleosome modifications
2006-03-06 | GSE3340 | GEO
Project description:This paper describes a simple and efficient walking method for constructing high resolution physical maps and discusses its applications to genome analysis. The method is an integration of three strategies: (1) use of a highly redundant library of 3Kb-long subclones; (2) construction of a multidimensional pool from the library; (3) direct application of a PCR (polymerase chain reaction)-based screening technique to the pooled library, with two PCR primers, one from the end of the subcloning vector and the other from the leading edge of the walk. This technique allows not only detection of each overlapping subclone but simultaneous determination of its orientation and the size of its overlap. The end of the subclone with the smallest overlap is sequenced and a primer is designed for the next step in the walk. Iteration of the screening procedure with minimum overlapping subclones results in completion of the high resolution map. Using this method, a 3Kb-resolution map was constructed from an 80Kb region of the bithorax complex of Drosophila melanogaster. The method is general enough to be applicable to DNA from other species, and simple enough to be automated.
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Project description:Active DNA demethylation in mammals involves TET-mediated iterative oxidation of 5-methylcytosine (5mC)/5-hydroxymethylcytosine (5hmC) and subsequent excision repair of highly oxidized cytosine bases 5-formylcytosine (5fC)/5-carboxylcytosine (5caC) by Thymine DNA glycosylase (TDG). However, quantitative and high-resolution analysis of active DNA demethylation activity remains challenging. Here we describe M.SssI methylase-assisted bisulfite sequencing (MAB-seq), a method that directly maps 5fC/5caC at single-base resolution. Genome-wide MAB-seq allows systematic identification of 5fC/5caC in Tdg-depleted embryonic stem cells, thereby generating a base-resolution map of active DNA demethylome. A comparison of 5fC/5caC and 5hmC distribution maps indicates that catalytic processivity of TET enzymes correlates with local chromatin accessibility. MAB-seq also reveals strong strand asymmetry of active demethylation within palindromic CpGs. Integrating MAB-seq with other base-resolution mapping methods enables quantitative measurement of cytosine modification states at key transitioning steps of active demethylation pathway, and reveals a regulatory role of 5fC/5caC excision repair in active DNA demethylation cascade. Analysis of 5fC/5caC excision repair-dependent active DNA demethylome by MAB-seq in mouse embryonic stem cells.
2014-11-02 | E-GEOD-62631 | biostudies-arrayexpress
Project description:Cytosine deaminases have important uses in the detection of epigenetic modifications and in genome editing. However, the range of applications of deaminases is limited by their substrate preference. To expand the toolkit of deaminases, we developed an in-vitro approach that bypasses a major hurdle with their severe toxicity in expression hosts. We screened 175 putative cytosine deaminases, primarily from bacteria, and found enzymes with strong activity on double- and single-stranded DNA in various sequence contexts, including some without any sequence constraints. We also found enzymes that do not deaminate modified cytosines. The remarkable diversity of cytosine deaminases opens new avenues for biotechnological and medical applications. As a demonstration, we developed a single-enzyme methylation sequencing (SEM-seq) method for 5-methylcytosine detection using a novel non-specific, modification-sensitive double-stranded DNA deaminase, MsddA. SEM-seq generated accurate base-resolution maps of 5-methylcytosine in human genome samples including cell free DNA and samples of 10 pg DNA, equivalent to single cell input. This simple and efficient protocol has the potential to allow high-throughput epigenome profiling of scarce biological material.
2023-10-05 | GSE233932 | GEO
Project description:Background: As costs decline, the size and scope of microarray experiments have increased. In multi-centre studies there is a need to ensure consistency of data pre-processing across centres. Similarly, in smaller scale studies the evolution of microarray platforms means that there is often a need to compare data generated on earlier microarrays to that generated on newer ones. It is important in such studies to ensure that platform-dependent biases are removed so that meta-analysis of different datasets can be performed reliably. In both these cases the optimal scenario is to have a small subset of samples repeated at each site or on each platform. These replicates can then be used to learn a relationship between probe intensities on the two platforms. Results: I introduce here a simple, linear-modelling-based method for normalizing data from multiple-platforms by using replicate hybridizations. A dataset of 20 rat liver samples is used as a benchmark. Eight samples are hybridized to two separate versions of Affymetrix microarrays, while the other 12 are hybridized to one, for a total of 28 arrays. Our linear modelling method removes platform bias as assessed using both unsupervised machine-learning and two-group statistical analyses. The method is computationally efficient and works well for data pre-processed by the GCRMA, RMA and MAS5 algorithms and using either default or alternative probe-mappings. The method is very stable towards the number of replicate samples used, with even two replicates greatly reducing platform-specific bias. Conclusions: A simple linear-modelling method can remove platform-specific bias independent of the pre-processing algorithm and ProbeSet-mapping used. This technique can readily be extended to multi-site experiments, and suggests the benefits of including a small number of replicate hybridizations in each new study as a normalization control. Twenty rats livers were processed, eight on both RAE230-A and RAE230-2 arrays, 8 on only RAE230-A arrays, and 4 on RAE230-2 arrays only.
2010-06-25 | E-GEOD-16458 | biostudies-arrayexpress
Project description:We have adapted the eXcision Repair-sequencing (XR-seq) method to generate single-nucleotide resolution dynamic repair maps of UV-induced cyclobutane pyrimidine dimers (CPDs) and (6-4) pyrimidine-pyrimidone photoproducts [(6-4)PPs] in the Saccharomyces cerevisiae genome. We find that these photoproducts are removed from the genome primarily by incisions 13-18 nucleotides 5’ and 6-7 nucleotides 3’ to the UV damage that generate 21-27 nt-long excision products. Analyses of the excision repair kinetics both in single genes and at the genome-wide level reveal strong transcription-coupled repair of the transcribed strand (TS) at early time points followed by predominantly non-transcribed strand (NTS) repair at later stages. We have also characterized the excision repair level as a function of transcription level. The availability of high-resolution and dynamic repair maps should aid in future repair and mutagenesis studies in this model organism.
2018-04-12 | GSE110621 | GEO