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Genome-wide CRISPR screen in standard growth conditions and stem cell conditions using primary low-passage KPf/fC pancreatic cancer cell lines

ABSTRACT: Genome-wide CRISPR screen in standard growth conditions and stem cell conditions using primary low-passage KPf/fC pancreatic cancer cell lines

PROVIDER: PRJNA473084 | ENA |

REPOSITORIES: ENA

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			Action	DRS
	SRR7217460.fastq.gz	Fastqsanger.gz
	SRR7217461.fastq.gz	Fastqsanger.gz
	SRR7217462.fastq.gz	Fastqsanger.gz
	SRR7217463.fastq.gz	Fastqsanger.gz
	SRR7217455.fastq.gz	Fastqsanger.gz

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Genome-wide CRISPR screen in standard growth conditions and stem cell conditions using primary low-passage KPf/fC pancreatic cancer cell lines

Project description:Purpose: The goal of this study is to identify novel genes required for the survival and proliferation of pancreatic cancer cells in standard culture conditions and in stem cell conditions Methods: Three independent primary pancreatic cancer cell lines were established from endstage Musashi2-reporter KPf/fC mice, and transduced with the genome-wide lentiCRISPR v2 library. Cells were grown under puromycin selection for 3 weeks (6 passages), then plated in non-adherent stem cell conditions in single-cell density for 1 week. Genomic DNA was isolated directly after library transduction, after selection in standard growth conditions, and after sphere growth in stem cell conditions. For sequencing, guide RNA's were amplified from total genomic DNA and barcoded for sequencing on an Illumina HiSeq2500 using V4 sequencing chemistry Conclusions: Our study represents the first genome-wide CRISPR screen identifying genetic vulnerabilities of KPf/fC cells in stem cell conditions using 3 biologically unique primary cell lines.

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Project description:Global Run-On has been performed in MLL2 F/F and FC/FC MEF cells. The aim of the experiment is to test how the lack of MLL2 affects nascent RNA transcription. Four samples were analyzed - 2 independent replicates. The experiments were performed using immortalized mouse embryonic fibroblasts (MEF) in which both MLL2 alleles were targeted by the loxp system (F/F cells). Tamoxifen treatment of the F/F cells for 24 hours results in the excision of both MLL2 alleles (FC/FC cells).

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Project description:Induced pluripotent stem cells (iPSCs) are a valuable resource for neurological disease-modeling and drug discovery, due to their ability to differentiate into neurons reflecting the genetics of the patient from which they are derived. iPSC-derived cultures, however, are highly variable due to differences in culture conditions. We investigated the effect of iPSC passage number on differentiation to optimize the generation of functional, mature sensory neurons (iPSC-dSNs). Three iPSC lines were differentiated into iPSC-dSNs at passage numbers within each of the following ranges: low (LP; 5-10), middle (MP; 20-26), and high (HP; 30-38). Morphology and pluripotency of the parent iPSCs were assessed prior to differentiation at each passage number. iPSC-dSNs were evaluated based on electrophysiological properties and expression of key neuronal markers. All iPSC lines displayed the same morphology and were similarly pluripotent across passage numbers. iPSC-dSNs were also morphologically comparable across passage numbers. However, the expression levels of neuronal markers and an analysis of sodium channel function indicated greater maturity in LP iPSC-dSNs. Our results demonstrate that lower passage numbers may be better suited for differentiation into peripheral sensory neurons. Further studies are warranted to elucidate factors that may contribute to the variability associated with iPSC passage number.

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