An optimized NGS sample preparation protocol for in vitro CRISPR screens

STAR Protocols

Charles M. Rudin¹  Triparna Sen²  Àlvaro Quintanal-Villalonga²  Fathema Uddin²  Corrin A. Wohlhieter³  John T. Poirier⁴ 

[1] Molecular Pharmacology Program, Memorial Sloan Kettering Cancer Center, New York, NY 10065, USA;Department of Medicine, Memorial Sloan Kettering Cancer Center, New York, NY 10065, USA;Graduate Program in Pharmacology, Weill Cornell Medicine, New York, NY 10021, USA;Perlmutter Cancer Center, New York University Langone Health, New York, NY 10016, USA;

关键词: Cancer;    High Throughput Screening;    Molecular Biology;    CRISPR;   

DOI  :  

来源: DOAJ

【 摘 要 】

Summary: This standardized protocol describes the preparation of PCR amplified and purified samples from human cell lines passaged and collected from CRISPR screening. High-quality samples can be used to perform next-generation sequencing (NGS) to uncover changes in sgRNA abundance from the timepoint at which library-transduced cells are selected to the timepoint when the screen is ended. Here, we describe proper calculation methods for library representation and show how to overcome potential issues often encountered by researchers.For complete information on the use and execution of this protocol, please refer to Wohlhieter et al. (2020).

【 授权许可】

Unknown