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 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 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.

scholarly journals Megakaryocyte Progenitor Cell Function is Enhanced Upon Aging Despite the Functional Decline of Aged Hematopoietic Stem Cells

2021 ◽  
Author(s):  
Donna M. Poscablo ◽  
Atesh K. Worthington ◽  
Stephanie Smith-Berdan ◽  
E. Camilla Forsberg
Keyword(s):  
Cell Function ◽  
Functional Decline ◽  
List Type ◽  
Hematopoietic Stem ◽  
Cell Numbers ◽  
Age Related ◽  
Genome Wide ◽  
Expansion Capacity ◽  
Old Mice

SUMMARYAge-related morbidity is associated with a decline in hematopoietic stem cell (HSC) function, but the mechanisms of HSC aging remain unclear. We performed heterochronic HSC transplants followed by quantitative analysis of cell reconstitution. While young HSCs outperformed old HSCs in young recipients, young HSCs unexpectedly failed to outcompete the old HSCs of aged recipients. Interestingly, despite substantial enrichment of megakaryocyte progenitors (MkPs) in old mice in situ and reported platelet (Plt) priming with age, transplanted old HSCs were deficient in reconstitution of all lineages, including MkPs and Plts. We therefore performed functional analysis of young and old MkPs. Surprisingly, old MkPs displayed unmistakably greater regenerative capacity compared to young MkPs. Transcriptome analysis revealed putative molecular regulators of old MkP expansion. Collectively, these data demonstrated that aging affects HSCs and megakaryopoiesis in fundamentally different ways: whereas old HSCs functionally decline, MkPs gain expansion capacity upon aging.HIGHLIGHTSFrequencies and total cell numbers of HSCs and MkPs were increased upon agingReconstitution deficit by old HSCs was observed by chimerism and absolute cell numbersYoung HSCs did not have competitive advantage over old HSCs in aged recipient miceOld MkPs display remarkable capacity to engraft, expand, and reconstitute plateletsAging is associated with changes in MkP genome-wide expression signatures

scholarly journals Influence of Casting Solvents on CO2/CH4 Separation Using Polysulfone Membranes

Membranes ◽  
2021 ◽  
Vol 11 (4) ◽  
pp. 286
Author(s):  
Roba M. Almuhtaseb ◽  
Ahmed Awadallah-F ◽  
Shaheen A. Al-Muhtaseb ◽  
Majeda Khraisheh
Keyword(s):  
High Efficiency ◽  
In Situ Synthesis ◽  
Separation Process ◽  
Polymeric Membranes ◽  
Gas Mixture ◽  
Manufacturing Cost ◽  
Binary Gas Mixture ◽  
Maximum Permeability ◽  
Polysulfone Membranes

Polysulfone membranes exhibit resistance to high temperature with low manufacturing cost and high efficiency in the separation process. The composition of gases is an important step that estimates the efficiency of separation in membranes. As membrane types are currently becoming in demand for CO2/CH4 segregation, polysulfone will be an advantageous alternative to have in further studies. Therefore, research is undertaken in this study to evaluate two solvents: chloroform (CF) and tetrahydrofuran (THF). These solvents are tested for casting polymeric membranes from polysulfone (PSF) to separate every single component from a binary gas mixture of CO2/CH4. In addition, the effect of gas pressure was conducted from 1 to 10 bar on the behavior of the permeability and selectivity. The results refer to the fact that the maximum permeability of CO2 and CH4 for THF is 62.32 and 2.06 barrer at 1 and 2 bars, respectively. Further, the maximum permeability of CF is 57.59 and 2.12 barrer at 1 and 2 bars, respectively. The outcome selectivity values are 48 and 36 for THF and CF at 1 bar, accordingly. Furthermore, the study declares that with the increase in pressure, the permeability and selectivity values drop for CF and THF. The performance for polysulfone (PSF) membrane that is manufactured with THF is superior to that of CF relative to the Robeson upper bound. Therefore, through the results, it can be deduced that the solvent during in-situ synthesis has a significant influence on the gas separation of a binary mixture of CO2/CH4.

scholarly journals Identification, Expression and Evolution of Short-Chain Dehydrogenases/Reductases in Nile Tilapia (Oreochromis niloticus)

2021 ◽  
Vol 22 (8) ◽  
pp. 4201
Author(s):  
Shuai Zhang ◽  
Lang Xie ◽  
Shuqing Zheng ◽  
Baoyue Lu ◽  
Wenjing Tao ◽  
...  
Keyword(s):  
Transcriptome Analysis ◽  
Tandem Duplication ◽  
Developmental Stages ◽  
Short Chain ◽  
Sexually Dimorphic ◽  
Physiological Processes ◽  
Genome Wide ◽  
Nile Tilapia Oreochromis Niloticus ◽  
Tandem Duplications

The short-chain dehydrogenases/reductases (SDR) superfamily is involved in multiple physiological processes. In this study, genome-wide identification and comprehensive analysis of SDR superfamily were carried out in 29 animal species based on the latest genome databases. Overall, the number of SDR genes in animals increased with whole genome duplication (WGD), suggesting the expansion of SDRs during evolution, especially in 3R-WGD and polyploidization of teleosts. Phylogenetic analysis indicated that vertebrates SDRs were clustered into five categories: classical, extended, undefined, atypical, and complex. Moreover, tandem duplication of hpgd-a, rdh8b and dhrs13 was observed in teleosts analyzed. Additionally, tandem duplications of dhrs11-a, dhrs7a, hsd11b1b, and cbr1-a were observed in all cichlids analyzed, and tandem duplication of rdh10-b was observed in tilapiines. Transcriptome analysis of adult fish revealed that 93 SDRs were expressed in more than one tissue and 5 in one tissue only. Transcriptome analysis of gonads from different developmental stages showed that expression of 17 SDRs were sexually dimorphic with 11 higher in ovary and 6 higher in testis. The sexually dimorphic expressions of these SDRs were confirmed by in situ hybridization (ISH) and qPCR, indicating their possible roles in steroidogenesis and gonadal differentiation. Taken together, the identification and the expression data obtained in this study contribute to a better understanding of SDR superfamily evolution and functions in teleosts.

Study on Mechanical Automation with Design of Rapid Milk Detector Based on Freezing Point

2013 ◽  
Vol 703 ◽  
pp. 282-286
Author(s):  
Ren Cai Zhang ◽  
Xiang Yu ◽  
Xing Ju Liu ◽  
Jin Hai Zhai ◽  
Zhen Wu Ning
Keyword(s):  
Integrated Circuit ◽  
Temperature Sensor ◽  
High Efficiency ◽  
Freezing Point ◽  
Electronic Control ◽  
Freezing Point Depression ◽  
Electronic Control System ◽  
Freezing Curve ◽  
Rack Mechanism

An efficient automated milk detector based on freezing point depression is designed. This detector shares characters of high efficiency and good stability with accuracy and automation. Its main parts include temperature sensor of IC (Integrated Circuit), pinion-rack mechanism and crank-rocker mechanism and electronic control system. Monitoring in-situ change of milk freezing curve and developing efficiency of sampling can be available by means of pinion-rack mechanism and IC temperature sensor mechatronics design. As a result, adulterating status of milk can be discriminated in a rapid and accurate and automated way. The detector may be employed to detect liquid foods other than milk as well.

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