Viral Packaging and Cell Culture for CRISPR-Based Screens
Name
Lander_Viral packaging.pdf
Size
139.59 KB
Format
Adobe PDF
Checksum (MD5)
965618242903f6b72b3e6d4b67440e9a
Author(s)
Wang, Tim
Lander, Eric Steven
Sabatini, David
Date Issued
March 2016
Journal
Cold Spring Harbor Protocols
Publisher
Cold Spring Harbor Laboratory Press
Citation
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.
Version
Author's final manuscript
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 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.
MIT Department
Massachusetts Institute of Technology. Department of Biology
Whitehead Institute for Biomedical Research
Koch Institute for Integrative Cancer Research at MIT
Terms of Use
Creative Commons Attribution-Noncommercial-Share Alike
Persistent DSpace Link
DOI of Published Version
https://doi.org/10.1101/pdb.prot090811
