scholarly journals An efficient targeted nuclease strategy for high-resolution mapping of DNA binding sites

2016 ◽  
Author(s):  
Peter J. Skene ◽  
Steven Henikoff
Keyword(s):  
Transcription Factor ◽  
High Resolution ◽  
Cost Effective ◽  
Specific Protein ◽  
Micrococcal Nuclease ◽  
Attractive Alternative ◽  
Dna Binding Sites ◽  
Resolution Mapping ◽  
Chromatin Profiling

AbstractWe describe Cleavage Under Targets and Release Using Nuclease (CUT&RUN), a chromatin profiling strategy in which antibody-targeted controlled cleavage by micrococcal nuclease releases specific protein-DNA complexes into the supernatant for paired-end DNA sequencing. Unlike Chromatin Immunoprecipitation (ChIP), which fragments and solubilizes total chromatin, CUT&RUN is performed in situ, allowing for both quantitative high-resolution chromatin mapping and probing of the local chromatin environment. When applied to yeast and human nuclei, CUT&RUN yielded precise transcription factor profiles while avoiding cross-linking and solubilization issues. CUT&RUN is simple to perform and is inherently robust, with extremely low backgrounds requiring only ~1/10th the sequencing depth as ChIP, making CUT&RUN especially cost-effective for transcription factor and chromatin profiling. When used in conjunction with native ChIP-seq and applied to human CTCF, CUT&RUN mapped directional long range contacts at high resolution. We conclude that in situ mapping of protein-DNA interactions by CUT&RUN is an attractive alternative to ChIP-seq.

scholarly journals An efficient targeted nuclease strategy for high-resolution mapping of DNA binding sites

eLife ◽  
2017 ◽  
Vol 6 ◽  
Author(s):  
Peter J Skene ◽  
Steven Henikoff
Keyword(s):  
Transcription Factor ◽  
High Resolution ◽  
Cost Effective ◽  
Specific Protein ◽  
Micrococcal Nuclease ◽  
Attractive Alternative ◽  
Dna Binding Sites ◽  
Resolution Mapping ◽  
Chromatin Profiling

We describe Cleavage Under Targets and Release Using Nuclease (CUT&RUN), a chromatin profiling strategy in which antibody-targeted controlled cleavage by micrococcal nuclease releases specific protein-DNA complexes into the supernatant for paired-end DNA sequencing. Unlike Chromatin Immunoprecipitation (ChIP), which fragments and solubilizes total chromatin, CUT&RUN is performed in situ, allowing for both quantitative high-resolution chromatin mapping and probing of the local chromatin environment. When applied to yeast and human nuclei, CUT&RUN yielded precise transcription factor profiles while avoiding crosslinking and solubilization issues. CUT&RUN is simple to perform and is inherently robust, with extremely low backgrounds requiring only ~1/10th the sequencing depth as ChIP, making CUT&RUN especially cost-effective for transcription factor and chromatin profiling. When used in conjunction with native ChIP-seq and applied to human CTCF, CUT&RUN mapped directional long range contact sites at high resolution. We conclude that in situ mapping of protein-DNA interactions by CUT&RUN is an attractive alternative to ChIP-seq.

scholarly journals A simple method for generating high-resolution maps of genome-wide protein binding

eLife ◽  
2015 ◽  
Vol 4 ◽  
Author(s):  
Peter J Skene ◽  
Steven Henikoff
Keyword(s):  
Transcription Factor ◽  
High Resolution ◽  
Rna Polymerase Ii ◽  
Nuclease Activity ◽  
Transcription Factor Binding ◽  
Micrococcal Nuclease ◽  
Simple Method ◽  
Factor Binding ◽  
Genome Wide ◽  
Resolution Mapping

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 (<xref ref-type="bibr" rid="bib14">Skene et al., 2014</xref>). 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.

High-resolution mapping of human chromosome 11 by in situ hybridization with cosmid clones

Science ◽  
1990 ◽  
Vol 247 (4938) ◽  
pp. 64-69 ◽  
Author(s):  
P Lichter ◽  
C. Tang ◽  
K Call ◽  
G Hermanson ◽  
G. Evans ◽  
...  
Keyword(s):  
In Situ Hybridization ◽  
High Resolution ◽  
Human Chromosome ◽  
Chromosome 11 ◽  
High Resolution Mapping ◽  
Resolution Mapping

scholarly journals A Cost-Effective In Situ Zooplankton Monitoring System Based on Novel Illumination Optimization

Sensors ◽  
2020 ◽  
Vol 20 (12) ◽  
pp. 3471
Author(s):  
Zhiqiang Du ◽  
Chunlei Xia ◽  
Longwen Fu ◽  
Nan Zhang ◽  
Bowei Li ◽  
...  
Keyword(s):  
High Resolution ◽  
Imaging System ◽  
Physiological State ◽  
Cost Effective ◽  
In Situ Monitoring ◽  
Measuring System ◽  
Target Area ◽  
Image Processing Algorithm ◽  
Led Array

A cost-effective and low-power-consumption underwater microscopic imaging system was developed to capture high-resolution zooplankton images in real-time. In this work, dark-field imaging was adopted to reduce backscattering and background noise. To produce an accurate illumination, a novel illumination optimization scheme for the light-emitting diode (LED) array was proposed and applied to design a lighting system for the underwater optical imaging of zooplankton. A multiple objective genetic algorithm was utilized to find the best location of the LED array, which resulted in the specific illumination level and most homogeneous irradiance in the target area. The zooplankton imaging system developed with the optimal configuration of LEDs was tested with Daphnia magna under laboratory conditions. The maximal field of view was 16 mm × 13 mm and the optical resolution was 15 μm. The experimental results showed that the imaging system developed could capture high-resolution and high-definition images of Daphnia. Subsequently, Daphnia individuals were accurately segmented and their geometrical characters were measured by using a classical image processing algorithm. This work provides a cost-effective zooplankton measuring system based on an optimization illumination configuration of an LED array, which has a great potential for minimizing the investment and operating costs associated with long-term in situ monitoring of the physiological state and population conditions of zooplankton.

High resolution mapping of overlapping cosmids by fluorescence in situ hybridization

Cytometry ◽  
1994 ◽  
Vol 15 (3) ◽  
pp. 193-198 ◽  
Author(s):  
Timothy W. Houseal ◽  
William R. Dackowski ◽  
Gregory M. Landes ◽  
Katherine W. Klinger
Keyword(s):  
In Situ Hybridization ◽  
High Resolution ◽  
Fluorescence In Situ Hybridization ◽  
High Resolution Mapping ◽  
Resolution Mapping
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