Lentiviral Barcoding Plasmid Generation

A 6.4 kb MluI-ClaI fragment was isolated from pLenti6.2/V5DEST (Invitrogen) and ligated to a linker comprising oligonucleotides 5'-CGATAACTGCAGAACCAATGCATTGGA-3' and 5'-CGCGTCCAATGCATTGGTTCTGCAGTTAT-3'. A library of MluI-PstI linkers was constructed using 24-bp Luminex DNA barcodes^{9 (link)} placed within oligonucleotides 5'-CGCGTXXXXXXXXXXXXXXXXXXXXXXXXCTGCA-3' and 5'-GxxxxxxxxxxxxxxxxxxxxxxxxA-3', where XXX...XXX includes the sense barcode sequence and xxx...xxx includes the antisense barcode sequence, and each of these linkers was individually ligated into the MluI-PstI backbone of the above vector to generate lentiviral barcoding plasmids. Lentivirus was generated from lentivral barcoding plasmids as previously described²⁴ using pCMV-dR8.2 dvpr and pCMV-VSVG packaging vectors in FuGENE6-transfected (Roche Corporation) HEK-293T cells; viral supernatant was collected after 72h, passed through a sterile 0.45μm syringe filter (VWR cat. 28144-007), and stored at −80°C.

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Yu C., Mannan A.M., Yvone G.M., Ross K.N., Zhang Y.L., Marton M.A., Taylor B.R., Crenshaw A., Gould J.Z., Tamayo P., Weir B.A., Tsherniak A., Wong B., Garraway L.A., Shamji A.F., Palmer M.A., Foley M.A., Winckler W., Schreiber S.L., Kung A.L, & Golub T.R. (2016). High-throughput identification of genotype-specific cancer vulnerabilities in mixtures of barcoded tumor cell lines. Nature biotechnology, 34(4), 419-423.

Publication 2016

pLenti6.2/V5DEST HEK-293T cells

293t cells Backbone Lentivirus Library Oligonucleotides Plasmids Sterile Syringe Vectors

Corresponding Organization :

Other organizations : Broad Institute, Dana-Farber Cancer Institute

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Variable analysis

independent variables

Linker comprising oligonucleotides 5'-CGATAACTGCAGAACCAATGCATTGGA-3' and 5'-CGCGTCCAATGCATTGGTTCTGCAGTTAT-3'
MluI-PstI linkers constructed using 24-bp Luminex DNA barcodes placed within oligonucleotides 5'-CGCGTXXXXXXXXXXXXXXXXXXXXXXXXCTGCA-3' and 5'-GxxxxxxxxxxxxxxxxxxxxxxxxA-3'

dependent variables

Not explicitly mentioned

control variables

PCMV-dR8.2 dvpr and pCMV-VSVG packaging vectors used in FuGENE6-transfected HEK-293T cells to generate lentivirus

positive controls

Not mentioned

negative controls

Not mentioned

Annotations

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