Abstract
This chapter details a compendium of protocols that collectively enable the reader to perform a pooled shRNA and/or CRISPR screen—with methods to identify and validate positive controls and subsequent hits; establish a viral titer in the cell line of choice; create and screen libraries, sequence strategies, and bioinformatics resources to analyze outcomes. Collectively, this provides an overarching resource from the start to finish of a screening project, making this technology possible in all laboratories.
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Acknowledgments
The authors are grateful to James Goldmeyer and Louise Baskin (Dharmacon GE) for technical discussions regarding library design.
The Johnstone laboratory (R.W.J.) is supported by research funding from the Leukaemia Foundation of Australia, The Kids Cancer Project, the Cancer Council Victoria, the National Health and Medical Research Council of Australia (NHMRC) and the Victorian Cancer Agency.
The Shortt laboratory (J.S.) is supported by funding from the Victorian Cancer Agency and Snowdome Foundation Fellowship. Cancer Council Victoria Venture Grant.
The Victorian Centre for Functional Genomics (K.J.S.) is funded by the Australian Cancer Research Foundation (ACRF), the Australian Phenomics Network (APN) through funding from the Australian Government’s National Collaborative Research Infrastructure Strategy (NCRIS) program and the Peter MacCallum Cancer Centre Foundation.
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Cluse, L.A. et al. (2018). A Comprehensive Protocol Resource for Performing Pooled shRNA and CRISPR Screens. In: Jenkins, B. (eds) Inflammation and Cancer. Methods in Molecular Biology, vol 1725. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-7568-6_17
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DOI: https://doi.org/10.1007/978-1-4939-7568-6_17
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