Viral Packaging and Cell Culture for CRISPR-Based Screens
Author(s)Wang, Tim; Lander, Eric Steven; Sabatini, David
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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 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. 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.
DepartmentMassachusetts Institute of Technology. Department of Biology; Whitehead Institute for Biomedical Research; Koch Institute for Integrative Cancer Research at MIT
Cold Spring Harbor Protocols
Cold Spring Harbor Laboratory Press
Wang, Tim, Eric S. Lander, and David M. Sabatini. “Viral Packaging and Cell Culture for CRISPR-Based Screens.” Cold Spring Harbor Protocols 2016.3 (2016): pdb.prot090811.
Author's final manuscript