Rapid creation and quantitative monitoring of high coverage shRNA libraries

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

doi:10.1038/nmeth.1330

Rapid creation and quantitative monitoring of high coverage shRNA libraries

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

Research output: Contribution to journal › Article › Academic › peer-review

Abstract

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.

Original language	English
Pages (from-to)	443-5
Number of pages	3
Journal	Nature Methods
Volume	6
Issue number	6
DOIs	https://doi.org/10.1038/nmeth.1330
Publication status	Published - Jun 2009

Keywords

Base Sequence
Gene Library
Humans
Molecular Sequence Data
Polymerase Chain Reaction
RNA, Messenger
Sequence Analysis, RNA
Journal Article
Research Support, N.I.H., Extramural
Research Support, Non-U.S. Gov't

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10.1038/nmeth.1330

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title = "Rapid creation and quantitative monitoring of high coverage shRNA libraries",

abstract = "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.",

keywords = "Base Sequence, Gene Library, Humans, Molecular Sequence Data, Polymerase Chain Reaction, RNA, Messenger, Sequence Analysis, RNA, Journal Article, Research Support, N.I.H., Extramural, Research Support, Non-U.S. Gov't",

author = "Bassik, \{Michael C\} and Lebbink, \{Robert Jan\} and Churchman, \{L Stirling\} and Ingolia, \{Nicholas T\} and Weronika Patena and LeProust, \{Emily M\} and Maya Schuldiner and Weissman, \{Jonathan S\} and McManus, \{Michael T\}",

year = "2009",

month = jun,

doi = "10.1038/nmeth.1330",

language = "English",

volume = "6",

pages = "443--5",

journal = "Nature Methods",

issn = "1548-7091",

publisher = "Nature Research",

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TY - JOUR

T1 - Rapid creation and quantitative monitoring of high coverage shRNA libraries

AU - Bassik, Michael C

AU - Lebbink, Robert Jan

AU - Churchman, L Stirling

AU - Ingolia, Nicholas T

AU - Patena, Weronika

AU - LeProust, Emily M

AU - Schuldiner, Maya

AU - Weissman, Jonathan S

AU - McManus, Michael T

PY - 2009/6

Y1 - 2009/6

N2 - 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.

AB - 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.

KW - Base Sequence

KW - Gene Library

KW - Humans

KW - Molecular Sequence Data

KW - Polymerase Chain Reaction

KW - RNA, Messenger

KW - Sequence Analysis, RNA

KW - Journal Article

KW - Research Support, N.I.H., Extramural

KW - Research Support, Non-U.S. Gov't

U2 - 10.1038/nmeth.1330

DO - 10.1038/nmeth.1330

M3 - Article

C2 - 19448642

SN - 1548-7091

VL - 6

SP - 443

EP - 445

JO - Nature Methods

JF - Nature Methods

IS - 6

ER -

Rapid creation and quantitative monitoring of high coverage shRNA libraries

Abstract

Keywords

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