Viral Packaging and Cell Culture for CRISPR-Based Screens.
Cold Spring Harb Protoc
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Abstract | This protocol describes how to perform the tissue culture and high-throughput sequencing library preparation for a CRISPR-based screen. First, pantropic lentivirus is prepared from a single guide RNA (sgRNA) plasmid pool and applied to the target cells. Following antibiotic selection and a harvest of the initial population, cells are then cultured under the desired screening condition(s) for 14 population doublings. The sgRNA barcode sequences integrated in the genomic DNA of each cell population are amplified and subject to high-throughput sequencing. Guidelines for downstream analysis of the sequencing data are also provided. |
Year of Publication | 2016
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Journal | Cold Spring Harb Protoc
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Volume | 2016
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Issue | 3
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Pages | pdb.prot090811
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Date Published | 2016 Mar 01
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ISSN | 1559-6095
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URL | |
DOI | 10.1101/pdb.prot090811
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PubMed ID | 26933250
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PubMed Central ID | PMC4804706
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Grant list | F31 CA189437 / CA / NCI NIH HHS / United States
R01 CA103866 / CA / NCI NIH HHS / United States
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