scholarly journals CUT&RUN: Targeted in situ genome-wide profiling with high efficiency for low cell numbers

2017 ◽  
Author(s):  
Peter J. Skene ◽  
Steven Henikoff
Keyword(s):  
Chromatin Immunoprecipitation ◽  
High Efficiency ◽  
Specific Protein ◽  
Micrococcal Nuclease ◽  
Quality Data ◽  
High Quality Data ◽  
Left Behind ◽  
Dna Accessibility ◽  
Genome Wide

SUMMARYCleavage Under Targets and Release Using Nuclease (CUT&RUN) is an epigenomic profiling strategy in which antibody-targeted controlled cleavage by micrococcal nuclease releases specific protein-DNA complexes into the supernatant for paired-end DNA sequencing. As only the targeted fragments enter into solution, and the vast majority of DNA is left behind, CUT&RUN has exceptionally low background levels. CUT&RUN outperforms the most widely-used Chromatin Immunoprecipitation (ChIP) protocols in resolution, signal-to-noise, and depth of sequencing required. In contrast to ChIP, CUT&RUN is free of solubility and DNA accessibility artifacts and can be used to profile insoluble chromatin and to detect long-range 3D contacts without cross-linking. Here we present an improved CUT&RUN protocol that does not require isolation of nuclei and provides high-quality data starting with only 100 cells for a histone modification and 1000 cells for a transcription factor. From cells to purified DNA CUT&RUN requires less than a day at the lab bench.

scholarly journals Targeted in situ genome-wide profiling with high efficiency for low cell numbers

2018 ◽  
Vol 13 (5) ◽  
pp. 1006-1019 ◽  
Author(s):  
Peter J Skene ◽  
Jorja G Henikoff ◽  
Steven Henikoff
Keyword(s):  
High Efficiency ◽  
Cell Numbers ◽  
Genome Wide

scholarly journals The Use of Quantitative Digital Pathology to Measure Proteoglycan and Glycosaminoglycan Expression and Accumulation in Healthy and Diseased Tissues

2020 ◽  
pp. 002215542095914
Author(s):  
A. Sally Davis ◽  
Mary Y. Chang ◽  
Jourdan E. Brune ◽  
Teal S. Hallstrand ◽  
Brian Johnson ◽  
...  
Keyword(s):  
Artificial Intelligence ◽  
Immune Cells ◽  
Quantitative Data ◽  
Digital Pathology ◽  
Quality Data ◽  
High Quality ◽  
Pathology Laboratory ◽  
High Quality Data ◽  
Hidden Patterns

Advances in reagents, methodologies, analytic platforms, and tools have resulted in a dramatic transformation of the research pathology laboratory. These advances have increased our ability to efficiently generate substantial volumes of data on the expression and accumulation of mRNA, proteins, carbohydrates, signaling pathways, cells, and structures in healthy and diseased tissues that are objective, quantitative, reproducible, and suitable for statistical analysis. The goal of this review is to identify and present how to acquire the critical information required to measure changes in tissues. Included is a brief overview of two morphometric techniques, image analysis and stereology, and the use of artificial intelligence to classify cells and identify hidden patterns and relationships in digital images. In addition, we explore the importance of preanalytical factors in generating high-quality data. This review focuses on techniques we have used to measure proteoglycans, glycosaminoglycans, and immune cells in tissues using immunohistochemistry and in situ hybridization to demonstrate the various morphometric techniques. When performed correctly, quantitative digital pathology is a powerful tool that provides unbiased quantitative data that are difficult to obtain with other methods.

scholarly journals BLISS: quantitative and versatile genome-wide profiling of DNA breaks in situ

2016 ◽  
Author(s):  
Winston X. Yan ◽  
Reza Mirzazadeh ◽  
Silvano Garnerone ◽  
David Scott ◽  
Martin W. Schneider ◽  
...  
Keyword(s):  
High Efficiency ◽  
Embryonic Stem ◽  
Dna Breaks ◽  
Drug Induced ◽  
Low Input ◽  
Tissue Sections ◽  
Genome Wide ◽  
Primary Mouse

AbstractWe present a method for genome-wide DNA double-strand Breaks (DSBs) Labeling In Situ and Sequencing (BLISS) which, compared to existing methods, introduces several key features: 1) high efficiency and low input requirement by in situ DSB labeling in cells or tissue sections directly on a solid surface; 2) easy scalability by performing in situ reactions in multi-well plates; 3) high sensitivity by linearly amplifying tagged DSBs using in vitro transcription; and 4) accurate DSB quantification and control of PCR biases by using unique molecular identifiers. We demonstrate the ability to use BLISS to quantify natural and drug-induced DSBs in low-input samples of cancer cells, primary mouse embryonic stem cells, and mouse liver tissue sections. Finally, we applied BLISS to compare the specificity of CRISPR-associated RNA-guided endonucleases Cas9 and Cpf1, and found that Cpf1 has higher specificity than Cas9. These results establish BLISS as a versatile, sensitive, and efficient method for genome-wide DSB mapping in many applications.

Complications of the Intragastric Balloon

2018 ◽  
Author(s):  
Ashok Menon ◽  
Haris A. Khwaja ◽  
Ariel Ortiz Lagardere ◽  
Manoel Galvao Neto ◽  
Jaime Ponce
Keyword(s):  
High Rate ◽  
Quality Data ◽  
Complication Rates ◽  
High Quality Data ◽  
Visceral Injury ◽  
Poor Weight Loss ◽  
Bariatric Patients ◽  
Treatment Of Obesity ◽  
Gastric Bubble

Intragastric balloons (IGB) have been used in the treatment of obesity for over 30 years. The first notable IGB device (the Garren Edwards Gastric Bubble) was withdrawn from the market due to patients’ poor weight loss and a high rate of complications. Several subsequent devices have been designed to address these shortcomings, but high-quality data are needed to compare complication rates among devices. Mortality across all IGB variants is extremely low compared to other bariatric procedures. Complications are mainly associated with visceral injury related to device insertion and retrieval, and with the presence or migration of an in-situ device, such as gastric ulceration and perforation, intolerance, gastrointestinal obstruction, and, rarely, acute pancreatitis. The incidence of postoperative venous thromboembolism (VTE) has not been extensively investigated, and it is not clear whether VTE is device-related or is due to increased VTE risk in bariatric patients.

scholarly journals E9: The Fine Resolution Powder Diffractometer (FIREPOD) at BER II

2017 ◽  
Vol 3 ◽  
Author(s):  
Alexandra Franz ◽  
Andreas Hoser
Keyword(s):  
Rietveld Analysis ◽  
Quality Data ◽  
Data Sets ◽  
Ambient Conditions ◽  
High Quality Data ◽  
Elastic Neutron ◽  
Powder Diffractometer ◽  
Structure Solution ◽  
Fine Resolution

The E9 (FIREPOD) is an upgraded fine resolution powder diffractometer for elastic neutron scattering, obtaining high quality data sets for Rietveld analysis, structure solution and phase analysis under ambient conditions as well as in situ at low / high temperatures, magnetic fields, gas pressure and various atmospheres.

scholarly journals AutoRELACS: Automated Generation And Analysis Of Ultra-parallel ChIP-seq

2020 ◽  
Author(s):  
L. Arrigoni ◽  
F. Ferrari ◽  
J. Weller ◽  
C. Bella ◽  
U. Bönisch ◽  
...  
Keyword(s):  
Chromatin Immunoprecipitation ◽  
Data Generation ◽  
Sample Collection ◽  
Dna Interactions ◽  
High Quality ◽  
Automated Generation ◽  
Genome Wide ◽  
Protein Dna Interactions ◽  
Biological Interpretation

ABSTRACTChromatin immunoprecipitation followed by sequencing (ChIP-seq) is a method used to profile protein-DNA interactions genome-wide. RELACS (Restriction Enzyme-based Labeling of Chromatin in Situ) is a recently developed ChIP-seq protocol that deploys a chromatin barcoding strategy to enable standardized and high-throughput generation of ChIP-seq data. The manual implementation of RELACS is constrained by human processivity in both data generation and data analysis. To overcome these limitations, we have developed AutoRELACS, an automated implementation of the RELACS protocol using the liquid handler Biomek i7 workstation. We match the unprecedented processivity in data generation allowed by AutoRELACS with the automated computation pipelines offered by snakePipes. In doing so, we build a continuous workflow that streamlines epigenetic profiling, from sample collection to biological interpretation. Here, we show that AutoRELACS successfully automates chromatin barcode integration, and is able to generate high-quality ChIP-seq data comparable with the standards of the manual protocol, also for limited amounts of biological samples.

scholarly journals Integrated Cryo-Correlative Microscopy for Targeted Structural Investigation In Situ

2021 ◽  
Vol 29 (6) ◽  
pp. 20-25
Author(s):  
Marit Smeets ◽  
Anna Bieber ◽  
Cristina Capitanio ◽  
Oda Schioetz ◽  
Thomas van der Heijden ◽  
...  
Keyword(s):  
Focused Ion Beam ◽  
Electron Tomography ◽  
Ion Beam ◽  
Building Blocks ◽  
Quality Data ◽  
Cellular Interaction ◽  
High Quality Data ◽  
Cryo Electron Tomography ◽  
Sample Transfer

Abstract:Cryo-electron tomography (cryo-ET) has the potential to revolutionize our understanding of the building blocks of life since it provides the unique opportunity to study molecules and membrane architectures in the context of cellular interaction. In particular, the combination of fluorescence imaging with focused ion beam (FIB) milling allows the targeting of specific structures in thick cellular samples by preparing thin lamellae that contain a specific fluorescence marker. This technique has conventionally been time-consuming, as it requires sample transfer to multiple microscopes and presents several technical challenges that currently limit its success. Here we describe METEOR, a FIB-integrated microscopy solution that streamlines the correlative cryo-ET workflow. It protects the sample from ice contamination by minimizing handling steps, thus increasing the likelihood of high-quality data. It also allows for monitoring of the milling procedure to ensure the molecule of interest is captured and can then be imaged by cryo-ET.

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