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Quantitative Analysis of Musashi2-reporter positive and Musashi2-reporter negative KPf/fC pancreatic cancer cell transcriptomes

ABSTRACT: Quantitative Analysis of Musashi2-reporter positive and Musashi2-reporter negative KPf/fC pancreatic cancer cell transcriptomes

PROVIDER: PRJNA473030 | ENA |

REPOSITORIES: ENA

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			Action	DRS
	SRR7216590.fastq.gz	Fastqsanger.gz
	SRR7216591.fastq.gz	Fastqsanger.gz
	SRR7216586.fastq.gz	Fastqsanger.gz
	SRR7216587.fastq.gz	Fastqsanger.gz
	SRR7216588.fastq.gz	Fastqsanger.gz

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Quantitative Analysis of Musashi2-reporter positive and Musashi2-reporter negative KPf/fC pancreatic cancer cell transcriptomes

Project description:Purpose: The goal of this study is to compare the transcriptome of pancreatic cancer stem cells (Musashi2-reporter positive) to pancreatic cancer non-stem cells (Musashi2-reporter negative). Methods: Musashi2-reporter mice contain a GFP-cassette knocked into the first exon of the endogenous Musashi2 locus; heterozygous mice are used to track cells expressing Musashi2. These mice were crossed to the KPf/fC (Ptf1a-Cre; KrasG12D; p53f/f) mouse model of pancreatic cancer. Tumors from endstage Musashi2-reporter KPf/fC mice were dissociated and FACS sorted for EpCAM+/GFP+ (Musashi2+ stem cells) and EpCAM+/GFP- (Musashi2- non-stem cells). For sequencing, mRNA libraries were generated from total RNA and sequenced with 50 basepair (bp) single end reads (SR50) to a depth of approximately 30 million reads per sample on an Illumina HiSeq2500 using V4 sequencing chemistry. Conclusions: Our study provides comparative transcriptomic data from primary isolated pancreatic cancer stem and non-stem cells in biologic replicates.

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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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