高覆盖率shRNA文库的快速构建与定量监测

Bassik Michael C, Lebbink Robert Jan, Churchman L Stirling, Ingolia Nicholas T, Patena Weronika, LeProust Emily M, Schuldiner Maya, Weissman Jonathan S, McManus Michael T

Department of Cellular and Molecular Pharmacology, California Institute for Quantitative Biomedical Research, Howard Hughes Medical Institute, University of California, San Francisco, California, USA.

Nat Methods. 2009 Jun;6(6):443-5. doi: 10.1038/nmeth.1330. Epub 2009 May 17.

Short hairpin RNA libraries are limited by low efficacy of many shRNAs and by off-target effects, which give rise to false negatives and false positives, respectively. Here we present a strategy for rapidly creating expanded shRNA pools (approximately 30 shRNAs per gene) that are analyzed by deep sequencing (EXPAND). This approach enables identification of multiple effective target-specific shRNAs from a complex pool, allowing a rigorous statistical evaluation of true hits.

短发夹RNA文库受到许多短发夹RNA低效率和脱靶效应的限制，这两种情况分别导致假阴性和假阳性。在此，我们提出一种策略，用于快速创建通过深度测序分析的扩展短发夹RNA文库（每个基因约30个短发夹RNA）（EXPAND）。这种方法能够从复杂文库中鉴定出多个有效的靶标特异性短发夹RNA，从而对真正的命中结果进行严格的统计学评估。