scholarly journals Simultaneous profiling of chromatin accessibility and methylation on human cell lines with nanopore sequencing

2018 ◽  
Author(s):  
Isac Lee ◽  
Roham Razaghi ◽  
Timothy Gilpatrick ◽  
Michael Molnar ◽  
Norah Sadowski ◽  
...  
Keyword(s):  
Cell Lines ◽  
Human Cell ◽  
Large Scale ◽  
Human Cancer ◽  
Chromatin Accessibility ◽  
Nanopore Sequencing ◽  
Human Cell Lines ◽  
Sequencing Data ◽  
Structural Variations ◽  
Genetic Changes

ABSTRACTUnderstanding how the genome and the epigenome work together to control gene transcription has applications in our understanding of diseases such as human cancer. In this study, we combine the ability of NOMe-seq to simultaneously evaluate CpG methylation and chromatin accessibility, with long-read nanopore sequencing technology, a method we call nanoNOMe. We generated >60Gb whole-genome nanopore sequencing data for each of four human cell lines (GM12878, MCF-10A, MCF-7, MDA-MB-231) including repetitive regions inaccessible by short read sequencing. Using the long reads, we find that we can observe phased methylation and chromatin accessibility, large scale pattern changes, and genetic changes such as structural variations from a single assay.

scholarly journals Simultaneous profiling of chromatin accessibility and methylation on human cell lines with nanopore sequencing

2020 ◽  
Vol 17 (12) ◽  
pp. 1191-1199
Author(s):  
Isac Lee ◽  
Roham Razaghi ◽  
Timothy Gilpatrick ◽  
Michael Molnar ◽  
Ariel Gershman ◽  
...  
Keyword(s):  
Cell Lines ◽  
Human Cell ◽  
Chromatin Accessibility ◽  
Nanopore Sequencing ◽  
Human Cell Lines

Large-Scale Protein Profiling in Human Cell Lines Using Antibody-Based Proteomics

2011 ◽  
Vol 10 (9) ◽  
pp. 4066-4075 ◽  
Author(s):  
Linn Fagerberg ◽  
Sara Strömberg ◽  
Adila El-Obeid ◽  
Marcus Gry ◽  
Kenneth Nilsson ◽  
...  
Keyword(s):  
Cell Lines ◽  
Human Cell ◽  
Large Scale ◽  
Protein Profiling ◽  
Human Cell Lines

scholarly journals MetaTropismDB: a database of organ-specific metastasis induced by human cancer cell lines in mouse models

Database ◽  
2020 ◽  
Vol 2020 ◽  
Author(s):  
Matteo Giulietti ◽  
Marco Bastianoni ◽  
Monia Cecati ◽  
Annamaria Ruzzo ◽  
Massimo Bracci ◽  
...  
Keyword(s):  
Mouse Models ◽  
Cell Lines ◽  
Cancer Cell ◽  
Human Cell ◽  
Human Cancer ◽  
Cancer Cell Lines ◽  
Human Cell Lines ◽  
Human Cancer Cell ◽  
Human Cancer Cell Lines ◽  
Organ Specific

Abstract The organotropism is the propensity of metastatic cancer cells to colonize preferably certain distant organs, resulting in a non-random distribution of metastases. In order to shed light on this behaviour, several studies were performed by the injection of human cancer cell lines into immunocompromised mouse models. However, the information about these experiments is spread in the literature. For each xenograft experiment reported in the literature, we annotated both the experimental conditions and outcomes, including details on inoculated human cell lines, mouse models, injection methods, sites of metastasis, organs not colonized, rate of metastasis, latency time, overall survival and the involved genes. We created MetaTropismDB, a freely available database collecting hand-curated data useful to highlight the mechanisms of organ-specific metastasis. Currently, it stores the results of 513 experiments in which injections of 219 human cell lines have been carried out in mouse models. Notably, 296 genes involved in organotropic metastases have been collected. This specialized database allows the researchers to compare the current results about organotropism and plan future experiments in order to identify which tumour molecular signatures establish if and where the metastasis will develop. Database URL:  http://www.introni.it/Metastasis/metastasis.html

scholarly journals Evaluation and Design of Genome-wide CRISPR/Cas9 Knockout Screens

2017 ◽  
Author(s):  
Traver Hart ◽  
Amy Tong ◽  
Katie Chan ◽  
Jolanda Van Leeuwen ◽  
Ashwin Seetharaman ◽  
...  
Keyword(s):  
Cell Lines ◽  
Human Cell ◽  
Viral Vectors ◽  
Human Cancer ◽  
Gene Knockout ◽  
Essential Genes ◽  
Human Cell Lines ◽  
Protein Coding ◽  
Mammalian Cell Lines ◽  
Genome Scale

AbstractThe adaptation of CRISPR/Cas9 technology to mammalian cell lines is transforming the study of human functional genomics. Pooled libraries of CRISPR guide RNAs (gRNAs), targeting human protein-coding genes and encoded in viral vectors, have been used to systematically create gene knockouts in a variety of human cancer and immortalized cell lines, in an effort to identify whether these knockouts cause cellular fitness defects. Previous work has shown that CRISPR screens are more sensitive and specific than pooled library shRNA screens in similar assays, but currently there exists significant variability across CRISPR library designs and experimental protocols. In this study, we re-analyze 17 genome-scale knockout screens in human cell lines from three research groups using three different genome-scale gRNA libraries, using the Bayesian Analysis of Gene Essentiality (BAGEL) algorithm to identify essential genes, to refine and expand our previously defined set of human core essential genes, from 360 to 684 genes. We use this expanded set of reference Core Essential Genes (CEG2), plus empirical data from six CRISPR knockout screens, to guide the design of a sequence-optimized gRNA library, the Toronto KnockOut version 3.0 (TKOv3) library. We demonstrate the high effectiveness of the library relative to reference sets of essential and nonessential genes as well as other screens using similar approaches. The optimized TKOv3 library, combined with the CEG2 reference set, provide an efficient, highly optimized platform for performing and assessing gene knockout screens in human cell lines.

scholarly journals Large-Scale Population Study of Human Cell Lines Indicates that Dosage Compensation is Virtually Complete

PLoS Genetics ◽  
2005 ◽  
Vol preprint (2007) ◽  
pp. e9
Author(s):  
Colette M Johnston ◽  
Frances L Lovell ◽  
Daniel A Leongamornlert ◽  
Barbara E. Stranger ◽  
Emmanouil T. Dermitzakis ◽  
...  
Keyword(s):  
Cell Lines ◽  
Population Study ◽  
Dosage Compensation ◽  
Human Cell ◽  
Large Scale ◽  
Human Cell Lines ◽  
Scale Population

scholarly journals A scalable strategy for high-throughput GFP tagging of endogenous human proteins

2016 ◽  
Vol 113 (25) ◽  
pp. E3501-E3508 ◽  
Author(s):  
Manuel D. Leonetti ◽  
Sayaka Sekine ◽  
Daichi Kamiyama ◽  
Jonathan S. Weissman ◽  
Bo Huang
Keyword(s):  
Cell Lines ◽  
Human Cell ◽  
Protein Function ◽  
Large Scale ◽  
Protein Complexes ◽  
Protein Localization ◽  
Human Cell Lines ◽  
Guide Rna ◽  
Human Genes ◽  
Endogenous Loci

A central challenge of the postgenomic era is to comprehensively characterize the cellular role of the ∼20,000 proteins encoded in the human genome. To systematically study protein function in a native cellular background, libraries of human cell lines expressing proteins tagged with a functional sequence at their endogenous loci would be very valuable. Here, using electroporation of Cas9 nuclease/single-guide RNA ribonucleoproteins and taking advantage of a split-GFP system, we describe a scalable method for the robust, scarless, and specific tagging of endogenous human genes with GFP. Our approach requires no molecular cloning and allows a large number of cell lines to be processed in parallel. We demonstrate the scalability of our method by targeting 48 human genes and show that the resulting GFP fluorescence correlates with protein expression levels. We next present how our protocols can be easily adapted for the tagging of a given target with GFP repeats, critically enabling the study of low-abundance proteins. Finally, we show that our GFP tagging approach allows the biochemical isolation of native protein complexes for proteomic studies. Taken together, our results pave the way for the large-scale generation of endogenously tagged human cell lines for the proteome-wide analysis of protein localization and interaction networks in a native cellular context.

scholarly journals Investigating the Selectivity of Novel Chalcones for Human Cancer Cells Compared to Non‐Cancerous Human Cell Lines

2019 ◽  
Vol 33 (S1) ◽  
Author(s):  
Christopher R.T. Stang ◽  
Laura R. Inbody ◽  
Mikaela C. Hickey ◽  
Ahmed H. Mohamed ◽  
Courtney A. Hollenbacher ◽  
...  
Keyword(s):  
Cancer Cells ◽  
Cell Lines ◽  
Human Cell ◽  
Human Cancer ◽  
Human Cell Lines ◽  
Human Cancer Cells

scholarly journals iCSDB: an integrated database of CRISPR screens

2020 ◽  
Vol 49 (D1) ◽  
pp. D956-D961
Author(s):  
Ahyoung Choi ◽  
Insu Jang ◽  
Heewon Han ◽  
Min-Seo Kim ◽  
Jinhyuk Choi ◽  
...  
Keyword(s):  
Cell Lines ◽  
Human Cell ◽  
High Throughput Screening ◽  
Large Scale ◽  
Target Genes ◽  
Human Cell Lines ◽  
Guide Rna ◽  
Functional Studies ◽  
Screening Experiments ◽  
Public Data

Abstract High-throughput screening based on CRISPR-Cas9 libraries has become an attractive and powerful technique to identify target genes for functional studies. However, accessibility of public data is limited due to the lack of user-friendly utilities and up-to-date resources covering experiments from third parties. Here, we describe iCSDB, an integrated database of CRISPR screening experiments using human cell lines. We compiled two major sources of CRISPR-Cas9 screening: the DepMap portal and BioGRID ORCS. DepMap portal itself is an integrated database that includes three large-scale projects of CRISPR screening. We additionally aggregated CRISPR screens from BioGRID ORCS that is a collection of screening results from PubMed articles. Currently, iCSDB contains 1375 genome-wide screens across 976 human cell lines, covering 28 tissues and 70 cancer types. Importantly, the batch effects from different CRISPR libraries were removed and the screening scores were converted into a single metric to estimate the knockout efficiency. Clinical and molecular information were also integrated to help users to select cell lines of interest readily. Furthermore, we have implemented various interactive tools and viewers to facilitate users to choose, examine and compare the screen results both at the gene and guide RNA levels. iCSDB is available at https://www.kobic.re.kr/icsdb/.

scholarly journals A scalable strategy for high-throughput GFP tagging of endogenous human proteins

2016 ◽  
Author(s):  
Manuel D. Leonetti ◽  
Sayaka Sekine ◽  
Daichi Kamiyama ◽  
Jonathan S. Weissman ◽  
Bo Huang
Keyword(s):  
Cell Lines ◽  
Human Cell ◽  
Large Scale ◽  
Human Proteome ◽  
Human Cell Lines ◽  
Functional Sequence ◽  
Human Genes ◽  
Human Proteins ◽  
The Way

AbstractA central challenge of the post-genomic era is to comprehensively characterize the cellular role of the ∼20,000 proteins encoded in the human genome. To systematically study protein function in a native cellular background, libraries of human cell lines expressing proteins tagged with a functional sequence at their endogenous loci would be very valuable. Here, using electroporation of Cas9/sgRNA ribonucleoproteins and taking advantage of a split-GFP system, we describe a scalable method for the robust, scarless and specific tagging of endogenous human genes with GFP. Our approach requires no molecular cloning and allows a large number of cell lines to be processed in parallel. We demonstrate the scalability of our method by targeting 48 human genes and show that the resulting GFP fluorescence correlates with protein expression levels. We next present how our protocols can be easily adapted for the tagging of a given target with GFP repeats, critically enabling the study of low-abundance proteins. Finally, we show that our GFP tagging approach allows the biochemical isolation of native protein complexes for proteomic studies. Together, our results pave the way for the large-scale generation of endogenously tagged human cell lines for the proteome-wide analysis of protein localization and interaction networks in a native cellular context.SIGNIFICANCE STATEMENTThe function of a large fraction of the human proteome still remains poorly characterized. Tagging proteins with a functional sequence is a powerful way to access function, and inserting tags at endogenous genomic loci allows the preservation of a near-native cellular background. To characterize the cellular role of human proteins in a systematic manner and in a native context, we developed a method for tagging endogenous human proteins with GFP that is both rapid and readily applicable at a genome-wide scale. Our approach allows studying both localization and interaction partners of the protein target. Our results pave the way for the large-scale generation of endogenously tagged human cell lines for a systematic functional interrogation of the human proteome.

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