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Open Access Open Badges Methodology article

Generation and analysis of a barcode-tagged insertion mutant library in the fission yeast Schizosaccharomyces pombe

Bo-Ruei Chen12, Devin C Hale235, Peter J Ciolek246 and Kurt W Runge12*

Author Affiliations

1 Department of Genetics, Case Western Reserve University School of Medicine, 10900 Euclid Avenue, Cleveland, OH, 44106, USA

2 Department of Molecular Genetics, Cleveland Clinic Lerner College of Medicine at CWRU, 9500 Euclid Avenue, NE20, Cleveland, OH, 44195, USA

3 John Carroll University, 20700 North Park Boulevard, University Heights, Ohio, 44118, USA

4 Miami University, 501 East High Street, Oxford, OH, 45056, USA

5 Current address: West Virginia School of Osteopathic Medicine, 400 North Lee Street, Lewisburg, WV, 24901, USA

6 Current address: College of Medicine, Ohio State University, 370 West 9th Avenue, Columbus, OH, 43210, USA

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BMC Genomics 2012, 13:161  doi:10.1186/1471-2164-13-161

Published: 3 May 2012

Additional files

Additional file 1:

Figure S1. A strategy to enrich for cells that have few copies of the ura4+ gene. Based on a hypothetical metabolic outcome of altered Ura4 protein levels and low concentrations of 5-FOA on cell survival, cells bearing few or many copies of ura4+ genes are expected to exhibit different sensitivities to 5-FOA. (A) In cells with multiple copies of the ura4+ gene, increased levels of Ura4 allow efficient conversion of low dose of 5-FOA to toxic 5-fluorouracil. These cells die in the medium supplied with a low concentration of 5-FOA (i.e. 0.1 g/l, data not shown). (B) In cells bearing a small number of copies of the ura4+ gene, endogenous orotidine-5-phosophate (orotidine-5P) may outcompete 5-FOA supplied in low concentrations as the preferred substrate of the limited amount of Ura4, prevent Ura4 from metabolizing 5-FOA to 5-fluorouracil and allow such cells to grow in medium with low 5-FOA.

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Additional file 2:

Table S1. Tetrad analysis and insertion site mapping of S. pombe barcoded insertion mutants.

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Additional file 3:

Figure S2. Characterization of insertion events by thermal asymmetric interlaced (TAIL)-PCR. (A) In mutants with a single or very few copies of insertion vectors, DNA fragments composed of partial insertion vector sequences and adjacent genomic DNA sequences could be amplified using vector-specific primers and a mixture of arbitrary degenerate (AD) primers. The nested vector-specific primers in each round of PCR reactions allowed for amplification of the final PCR products with increasing specificity. (B) In the event of tandem integration of multiple copies of the insertion vector, TAIL-PCR often resulted in amplification within insertion vectors due to additional binding sites for AD primers in the additional copies of insertion vectors. When vectors were integrated in head-to-head orientation, vector-specific primers could bind both strands of the repetitive insertion vector DNA and only amplify the vector sequences. (C) Mitochondrial DNA was found co-integrated with the insertion vector into the genome in some mutants. In such mutants, TAIL-PCR could only amplify DNA sequences corresponding to the insertion vector and mitochondrial DNA, which might result from additional binding site for AD primers in mitochondrial DNA.

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Additional file 4:

Figure S3. Characterization of insertion events by splinkerette PCR. In splinkerette PCR, genomic DNA is first digested with restriction enzymes that do not cut or cut very few times in the insertion vector (e.g. Xba I and Spe I, or Bcl I and Bgl II). Double strand splinkerette adaptor DNA can then be ligated to the digested genomic DNA with compatible overhangs. The resulting products, genomic fragments flanked by splinkerette and insertion vector DNA, can be amplified by PCR using primers on splinkerette (solid arrows) and the insertion vector (dashed arrows).

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Additional file 5:

Table S2. Insertion events with both insertion-genomic junctions characterized.

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Additional file 6:

Potential modes of integration of non-homologous DNA in S. pombe genome. After being transfected into S. pombe cells, non-homologous DNA could be integrated into the genome as a single copy. In some cases, the ends of non-homologous DNA may be first deleted by nucleolytic activities in cells and then ligated to other linear DNA fragments in head-to-tail, heat-to-head or tail-to-tail orientations before being integrated in the genome.

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Additional file 7:

Table S3. The amino acid and nucleobase supplements in the minimum medium + YC – uracil.

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Additional file 8:

Table S4. Oligonucleotides used in this study.

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Additional file 9:

pInsertion-ura4. Annotated sequence of pInsertion-ura4.

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Additional file 10:

pLox66. Annotated sequence of pLox66.

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