shRNA Core Facility

Libraries

Libraries Available in the shRNA Core Facility

The following table summarizes the gene-modulation libraries available in the shRNA Core Facility. For more detailed information, follow the links or scroll down the page.

  shRNA Libraries  CRISPR Libraries  ORF and cDNA Libraries 
   
Arrayed Libraries

For individual RNAi reagents and custom arrayed screening sets 
 
Pooled Libraries

For genome-scale selection-based screens 
 
CRISPR Libraries

For selection-based knockout screens 
 
ORF Libraries

In expression-ready lentiviral vectors 
 
cDNA Clone Libraries 
Human  TRC Genome-Wide shRNA Collection

GIPZ Lentiviral shRNAmir Library 
Decode RNAi-GIPZ Whole Genome Pooled Screening Library

DECIPHER Pooled Lentiviral shRNA Libraries 
Human Lentiviral sgRNA Libraries  Precision LentiORF Library

hORFeome V8.1 Library 
Mammalian Genome Collection 
Mouse  TRC Genome-Wide shRNA Collection

GIPZ Lentiviral shRNAmir Library 
DECIPHER Pooled Lentiviral shRNA Libraries      Mammalian Genome Collection 
 Rat  Partial coverage from above libraries due to homology to human and mouse         

  

TRC Libraries

pLKO.1The TRC Genome-Wide shRNA Collections for human and mouse were developed by The RNA Consortium.

  • shRNA design: Simple hairpin
  • Vector: pLKO.1 lentiviral vector. A full vector map and sequence are available in this document from the Broad Institute's Public TRC Portal.
  • Promoter: U6 (Pol III promoter)
  • Marker: None
  • Antibiotic resistance: Puromycin
  • Human library coverage:
    • Approximately 15,000 genes
    • Average of about 5 shRNAs per gene
     
  • Mouse library coverage:
    • Approximately 15,000 genes
    • Average of about 5 shRNAs per gene
     
  • Rat coverage (due to homology to human and mouse):
    • Over 10,000 genes
    • Average of about 2 shRNAs per gene
     
  • Clones are stored in the library as bacterial cultures of E. coli DH5α. 

References:

  1. Root DE, Hacohen N, Hahn WC, Lander ES, Sabatini DM. (2006) Genome-scale loss-of-function screening with a lentiviral RNAi library. Nature Methods 3:715.
  2. Moffat J, Grueneberg DA, Yang X, Kim SY, Kloepfer AM, Hinkle G, Piqani B, Eisenhaure TM, Luo B, Grenier JK, Carpenter AE, Foo SY, Stewart SA, Stockwell BR, Hacohen N, Hahn WC, Lander ES, Sabatini DM, Root DE. (2006) A lentiviral RNAi library for human and mouse genes applied to an arrayed viral high-content screen. Cell 124:1283.

 


 

 

GIPZ Libraries

pGIPZThe GIPZ Lentiviral shRNAmir Libraries were developed by Open Biosystems (Thermo Scientific) in collaboration with Greg Hannon (CSHL) and Steve Elledge (Harvard).

 

  • shRNA design: shRNAmir. The shRNA hairpin is placed in the context of the mir-30 sequence.
  • Vector: pGIPZ lentiviral vector. Details are available at the Open Biosystems website; a vector map and sequence are available here.
  • Promoter: CMV (Pol II promoter)
  • Marker: GFP
  • Antibiotic resistance: Puromycin
  • Human library coverage:
    • Approximately 18,000 genes
    • Average of about 5 shRNAs per gene
     
  • Mouse library coverage:
    • Over 16,000 genes
    • Average of 3-4 shRNAs per gene
     
  • Rat coverage (due to homology to human and mouse):
    • Over 10,000 genes
    • Average of about 2 shRNAs per gene
     
  • Clones are stored in the library as bacterial cultures of E. coli PrimePlus a version of JM109 modified for increased stability.

Reference:

  1. Silva JM, Li MZ, Chang K, Ge W, Golding MC, Rickles RJ, Siolas D, Hu G, Paddison PJ, Schlabach MR, Sheth N, Bradshaw J, Burchard J, Kulkarni A, Cavet G, Sachidanandam R, McCombie WR, Cleary MA, Elledge SJ, Hannon GJ. (2005) Second-generation shRNA libraries covering the mouse and human genomes. Nature Genetics 11:1281.

 


 

 

Decode Pooled shRNA Library

The Decode RNAi-GIPZ Whole Genome Pooled Screening Library from Open Biosystems (Thermo Scientific) is designed for use in selection-based screening, with hit identification by next-generation sequencing of the hairpin region.

 

  • shRNA design: shRNAmir. The shRNA hairpin is placed in the context of the mir-30 sequence.
  • Vector: pGIPZ lentiviral vector. Details are available at the Open Biosystems website; a vector map and sequence are available here.
  • Promoter: CMV (Pol II promoter)
  • Marker: GFP
  • Antibiotic resistance: Puromycin
  • Human pooled library coverage:
    • Approximately 50,000 clones targeting human genes, spread across 7 separate pools. Each pool targets approximately 6,000 genes, with some overlap between pools. The entire set targets over 20,000 genes, with each targeted by 2-3 shRNAs on average. 
     

Reference:

  1. Strezoska Z, Licon A, Haimes J, Spayd KJ, Patel KM, Sullivan K, Jastrzebski K, Simpson KJ, Leake D, van Brabant Smith A, Vermeulen A. (2012) Optimized PCR conditions and increased shRNA fold representation improve reproducibility of pooled shRNA screens. PLoS ONE 7:e42341.

  


 

 

DECIPHER Pooled shRNA Library

pRSI12 vector The DECIPHER Pooled Lentiviral shRNA Libraries from Cellecta is designed for use in selection-based screening, with hit identification by next-generation sequencing of a unique barcode sequence incorporated into each clone.

 

  • shRNA design: Simple hairpin
  • Vector: pRSI12 lentiviral vector. See details here.
  • Promoter: U6 (Pol III promoter)
  • Marker: RFP
  • Antiobiotic resistance: Puromycin
  • Human pooled library coverage:
    • Module 1 (~5,000 targets, 27,500 shRNAs)
    • Module 2 (~5,400 targets, 27,500 shRNAs)
    • Module 3 (~5,000 targets, 27,500 shRNAs)
     
  • Mouse pooled library coverage:
    • Module 1 (~5,000 targets, 27,500 shRNAs)
    • Module 2 (~5,000 targets, 27,500 shRNAs) 
     

 



 

CRISPR Pooled Libraries

pLX-sgRNAThe Human Lentiviral sgRNA Libraries, developed by the David Sabatini and Eric Lander labs (MIT/Broad Institute), are designed for use in selection-based screening, with hit identification by next-generation sequencing of the sgRNA.

 

  • Vectors: 2-vector system — pCW-Cas9, expressing Cas9 from an inducible promoter, snd pLX-sgRNA, expressing the sgRNA constitutively.
  • Promoters: Doxycycline-inducible Tet ON promoter for Cas9, U6 promoter for sgRNA
  • Marker: Cas9 tagged with FLAG; no marker on sgRNA vector
  • Antibiotic resistance: Puromycin on Cas9 vector, blasticidin on sgRNA vector
  • Design: 10 sgRNAs per gene
  • Gene coverage:
    • Kinase library: 507 genes
    • Cell cycle library: 983 genes
    • Nuclear protein library: 3,733 genes
     

 

Reference:

  1. Wang T, Wei JJ, Sabatini DM, Lander DS. (2014) Genetic screens in human cells using the CRISPR-Cas9 system.Science 343:80.

 



Precision LentiORF library

pLOCThe Human Precision LentiORF Library from Open Biosystems (Thermo Scientific) is an expression-ready collection of open reading frames, generated using content from the sequence-validated ORFeome Collaboration collection.

 

  • Vector: pLOC lentiviral expression vector. Vector details are available here.
  • Promoter: CMV (Pol II promoter)
  • Marker: GFP
  • Antibiotic resistance: Blasticidin
  • Human library coverage: ~6,000 genes

 



 

 

hORFeome V8.1 Library

pLX304The hORFeome V8.1 Library was developed at the Broad Institute.

 

  • Vector: pLX304 lentiviral expression vector. Vector details are available here.
  • Promoter: CMV (Pol II promoter)
  • Marker: None. All ORFs are tagged with the V5 epitope.
  • Antibiotic resistance: Blasticidin
  • Human library coverage: ~9,700 genes

 

Reference:

  1. Yang et al. (2010) A public genome-scale lentiviral expression library of human ORFs. Nature Methods 8:659.

 



 

 

Mammalian Genome Collection

 

The Mammalian Genome Collection was the result of a multi-institute effort based at the NIH, with the goal of producing at least one sequence-validated full-length clone for each known human and mouse gene.

 

  • Vector: The clones in this collection are in a variety of vectors, some of which are mammalian expression vectors (but not lentiviral vectors). The most frequent vectors used are pCMV-SPORT6pCR4-TOPOpOTB7, and pCR-BluntII-TOPO, but many others are also represented in the collection.
  • Human library coverage: ~17,000 genes
  • Mouse library coverage: ~14,500 genes

 

Reference:

  1. Zuber et al. (2009) The completion of the Mammalian Genome Collection (MGC). Genome Research 19:2324.
 

 

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