Viral Packaging and Cell Culture for CRISPR-Based Screens
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 popul...
| Main Authors: | , , |
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| Other Authors: | |
| Format: | Article |
| Language: | en_US |
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Cold Spring Harbor Laboratory Press
2016
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| Online Access: | http://hdl.handle.net/1721.1/105364 https://orcid.org/0000-0002-4227-5163 https://orcid.org/0000-0002-1446-7256 |
| _version_ | 1826191985776525312 |
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| author | Wang, Tim Lander, Eric Steven Sabatini, David |
| author2 | Massachusetts Institute of Technology. Department of Biology |
| author_facet | Massachusetts Institute of Technology. Department of Biology Wang, Tim Lander, Eric Steven Sabatini, David |
| author_sort | Wang, Tim |
| collection | MIT |
| description | 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. |
| first_indexed | 2024-09-23T09:04:05Z |
| format | Article |
| id | mit-1721.1/105364 |
| institution | Massachusetts Institute of Technology |
| language | en_US |
| last_indexed | 2024-09-23T09:04:05Z |
| publishDate | 2016 |
| publisher | Cold Spring Harbor Laboratory Press |
| record_format | dspace |
| spelling | mit-1721.1/1053642022-09-30T13:14:47Z Viral Packaging and Cell Culture for CRISPR-Based Screens Wang, Tim Lander, Eric Steven Sabatini, David Massachusetts Institute of Technology. Department of Biology Whitehead Institute for Biomedical Research Koch Institute for Integrative Cancer Research at MIT Wang, Tim Lander, Eric Steven Sabatini, David 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. 2016-11-18T18:38:57Z 2016-11-18T18:38:57Z 2016-03 Article http://purl.org/eprint/type/JournalArticle 1940-3402 1559-6095 http://hdl.handle.net/1721.1/105364 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. https://orcid.org/0000-0002-4227-5163 https://orcid.org/0000-0002-1446-7256 en_US http://dx.doi.org/10.1101/pdb.prot090811 Cold Spring Harbor Protocols Creative Commons Attribution-Noncommercial-Share Alike http://creativecommons.org/licenses/by-nc-sa/4.0/ application/pdf Cold Spring Harbor Laboratory Press PMC |
| spellingShingle | Wang, Tim Lander, Eric Steven Sabatini, David Viral Packaging and Cell Culture for CRISPR-Based Screens |
| title | Viral Packaging and Cell Culture for CRISPR-Based Screens |
| title_full | Viral Packaging and Cell Culture for CRISPR-Based Screens |
| title_fullStr | Viral Packaging and Cell Culture for CRISPR-Based Screens |
| title_full_unstemmed | Viral Packaging and Cell Culture for CRISPR-Based Screens |
| title_short | Viral Packaging and Cell Culture for CRISPR-Based Screens |
| title_sort | viral packaging and cell culture for crispr based screens |
| url | http://hdl.handle.net/1721.1/105364 https://orcid.org/0000-0002-4227-5163 https://orcid.org/0000-0002-1446-7256 |
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