Hahn TP53 Mutagenesis Library
(Pooled Library #113569)
The TP53 Mutagenesis by Integrated TilEs (MITE) library was designed for the characterization of all possible p53 mutations. The pooled lentiviral plasmid library contains ~8,000 single amino acid substitution and nonsense mutant variants of p53 (~25,000 codon variants). The backbone vector constitutively expresses p53 cDNA in human cells under the control of the EF1alpha promoter and harbors a puromycin resistance cassette for selection of infected cells.
Using this library, the Hahn lab performed screens to enrich for mutant p53 variants that display dominant-negative activity, loss-of-function, or wild-type-like function in A549 human cancer cells. They quantified variant representation by Nextera sample preparation followed by Illumina sequencing and data deconvolution, and subsequently determined variant enrichment and depletion in each screen.
Modifications: This backbone is a derivative of pLEX_307. The shorter fragment of NheI/MluI digestion of pLEX_307 was replaced with a fragment that contains multiple restriction cloning sites, and lacks Gateway att sites/ccdB/cmR. The TP53MITE library was cloned into pMT_BRD025 via the NheI/MluI sites.
|TP53 MITE Library
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p53 variants8,258 amino acid (up to 24,823 codon)
This library is delivered as suspended DNA in a microcentrifuge tube on blue ice. The tube's contents will not necessarily be frozen. For best results, minimize freeze-thaws.
To amplify this library, use at least 80 ng plasmid DNA library and 80 ul of Stbl4 competent cells (number of variants divided by 100), to obtain 1000x coverage.
Library representation was assessed by sequencing the plasmid DNA (pDNA) library using the Illumina Nextera XT method. Sequencing library prep protocols are available from Illumina. The resulting PCR products were cleaned by AMPure XP, using 1.8X volume of AMPure XP, and eluted with 52.5uL resuspension buffer. Quantification of the recovered PCR samples was performed by Qubit, and the samples were pooled by equal mass into a single tube and sequenced using Illumina NGS platforms. The Hahn lab typically performed 150nt paired-end reads, and the Illumina index reads, i5 and i7, are both 8nt long.
Since each variant differs from WT at a single codon, the sequencing reads are predominately WT. Thus, to quantify the relative abundance of the 8000 variants, a large number of reads are needed; please see the associated publication for details. The lab recommends using ORFcall software for sequencing data analysis.
These pooled libraries were created by your colleagues. Please acknowledge the Principal Investigator, cite the article in which the plasmids were described, and include Addgene in the Materials and Methods of your future publications.
For your Materials & Methods section:TP53 MITE library was a gift from William Hahn and David Root (Addgene #113569)
For your References section:Mutational processes shape the landscape of TP53 mutations in human cancer. Giacomelli AO, Yang X, Lintner RE, McFarland JM, Duby M, Kim J, Howard TP, Takeda DY, Ly SH, Kim E, Gannon HS, Hurhula B, Sharpe T, Goodale A, Fritchman B, Steelman S, Vazquez F, Tsherniak A, Aguirre AJ, Doench JG, Piccioni F, Roberts CWM, Meyerson M, Getz G, Johannessen CM, Root DE, Hahn WC. Nat Genet. 2018 Oct;50(10):1381-1387. doi: 10.1038/s41588-018-0204-y. PubMed 30224644