Format

Send to

Choose Destination
Cold Spring Harb Protoc. 2016 Mar 1;2016(3):pdb.prot090811. doi: 10.1101/pdb.prot090811.

Viral Packaging and Cell Culture for CRISPR-Based Screens.

Author information

1
Department of Biology, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139; Whitehead Institute for Biomedical Research, Cambridge, Massachusetts 02142; Broad Institute of MIT and Harvard, Cambridge, Massachusetts 02142; David H. Koch Institute for Integrative Cancer Research at MIT, Cambridge, Massachusetts 02139; Howard Hughes Medical Institute, Department of Biology, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139;
2
Department of Biology, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139; Broad Institute of MIT and Harvard, Cambridge, Massachusetts 02142; Department of Systems Biology, Harvard Medical School, Boston, Massachusetts 02115.

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.

PMID:
26933250
PMCID:
PMC4804706
DOI:
10.1101/pdb.prot090811
[Indexed for MEDLINE]
Free PMC Article

Supplemental Content

Full text links

Icon for HighWire Icon for PubMed Central
Loading ...
Support Center