Protocol Viral Packaging and Cell Culture for CRISPR-Based Screens Tim Wang, 1,2,3,4,5 Eric S. Lander, 1,3,6,7,8 and David M. Sabatini 1,2,3,4,5,7,8 1 Department of Biology, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139; 2 Whitehead Institute for Biomedical Research, Cambridge, Massachusetts 02142; 3 Broad Institute of MIT and Harvard, Cambridge, Massachusetts 02142; 4 David H. Koch Institute for Integrative Cancer Research at MIT, Cambridge, Massachusetts 02139; 5 Howard Hughes Medical Institute, Department of Biology, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139; 6 Department of Systems Biology, Harvard Medical School, Boston, Massachusetts 02115 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. MATERIALS It is essential that you consult the appropriate Material Safety Data Sheets and your institution’s Environmental Health and Safety Office for proper handling of equipment and hazardous materials used in this protocol. RECIPES: Please see the end of this protocol for recipes indicated by <R>. Additional recipes can be found online at http://cshprotocols.cshlp.org/site/recipes. Reagents Agarose gel (1.0%) Cell-culture medium appropriate for target cells Dulbecco’s Modified Eagle Medium (DMEM), high-glucose, GlutaMAX Supplement (Gibco 10566-016) Ethidium bromide Fetal bovine serum (inactivated) (Sigma-Aldrich F4135) Gel Extraction Kit (QIAGEN 28704) Human embryonic kidney (HEK) 293 T cells (ATCC CRL-3216) LB-ampicillin agar plates <R> 7 These authors contributed equally to this work 8 Correspondence: lander@broadinstitute.org; sabatini@wi.mit.edu © 2016 Cold Spring Harbor Laboratory Press Cite this protocol as Cold Spring Harb Protoc; doi:10.1101/pdb.prot090811 289 Cold Spring Harbor Laboratory Press at MASS INST OF TECHNOLOGY on March 7, 2016 - Published by http://cshprotocols.cshlp.org/ Downloaded from