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| Status |
Public on Jan 01, 2012 |
| Title |
Ty1 insertions in diploid yeast |
| Sample type |
SRA |
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| Source name |
yeast cells
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| Organism |
Saccharomyces cerevisiae |
| Characteristics |
strain: BY348
tip-seq: Ty1 flanking regions
cell type: diploid
genotype: BY348 (MATα his3Δ200 ura3-167 GAL+) containing plasmid pJEF2365
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| Growth protocol |
The strain used for the sequencing experiment was BY348 (MATα his3Δ200 ura3-167 GAL+) containing plasmid pJEF2365. pJEF2365 is a derivative of pVIT41 (Lauermann et al, 1997) with a higher performing 2 micron origin fragment derived from pJEF1562 (Monokian et al, 1994). The Ty1 element in this plasmid is tagged with a sequence called ssb (Fig. 1A). The media used were YPD, SC–Ura containing either 2% glucose (glu), 2% galactose, (gal) or 1% raffinose (raf), and SC+ 5-Foa (fluoro-orotic acid). Media were prepared as described (Rose et al, 1990, Smith et al, 1997).
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
Twelve individual colonies of strain BY348 were grown on SC–Ura glucose plates grown at 30˚C. These were used as the starting material for 96 independent galactose inductions to minimize jackpot effects. The colonies were picked into 5 mL SC–Ura 1% Raf and grown at 30˚C overnight. For each of the 12 cultures, corresponding to one column of a 96-deep well plate, the following were added to each well: a sterile 3 mm glass bead, 200 µL of the above inoculum and 1 mL SC–Ura gal. The plate was covered with Air Pore tape and grown at 22˚C at 240 rpm for 48 hours in a refrigerated air shaker. 1150 µL were removed from each well and replaced with 1150 µL of YPD and grown at 30˚C overnight with shaking to allow plasmid loss. 1150 µL were removed from each well and replaced with 1150 µL of SC + 1 mg/mL 5-Foa medium and grown overnight with shaking to select for plasmid free cells. The 8 cultures in each column were pooled to make a DNA preparation as described (Boeke et al, 1985). The DNA was then extracted three times with phenol/chloroform/isoamyl alcohol, and ethanol precipitated. Custom Illumina adapters were ordered as duplexes from IDT (top strand oligonucleotide;, 5’-pTAGTCCCTTAAGCGGAG-NH2; bottom strand oligonucleotide, 5’-GTAATACGACTCACTATAGGGCTCCGCTTAAGGGAC). 2 µG gDNA was digested to completion with 60 U MseI and 120 U BglII in a 100 µL reaction overnight and purified on a QiaQuick column (Qiagen). MseI-compatible adaptors were ligated overnight at 16˚C and heat inactivated at 70˚ for 15 min. Custom Illumina adapters were ordered as duplexes from IDT (top strand oligonucleotide;, 5’-pTAGTCCCTTAAGCGGAG-NH2; bottom strand oligonucleotide, 5’-GTAATACGACTCACTATAGGGCTCCGCTTAAGGGAC). 2 µG gDNA was digested to completion with 60 U MseI and 120 U BglII in a 100 µL reaction overnight and purified on a QiaQuick column (Qiagen). MseI-compatible adaptors were ligated overnight at 16˚C and heat inactivated at 70˚ for 15 min.
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| Library strategy |
OTHER |
| Library source |
genomic |
| Library selection |
other |
| Instrument model |
Illumina Genome Analyzer IIx |
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| Data processing |
flowcell62_lanedip.filter.R.clones.sequences; genome build: S288c, SGD 2/2009
We considered only those Illumina reads that started with the Ty1 LTR, allowing one “N” mismatch. After trimming sequence not derived from the yeast genome, the reads were aligned to the yeast genome using Bowtie (Langmead et al, 2009), using a seed of 20bp. Only uniquely and perfectly mapping reads were retained; reads mapping to identical genomic positions were counted only once to avoid biases due to PCR or other amplification artifacts.
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| Submission date |
Nov 28, 2011 |
| Last update date |
May 15, 2019 |
| Contact name |
Sarah Wheelan |
| Organization name |
Johns Hopkins University School of Medicine
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| Department |
Oncology
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| Street address |
550 N. Broadway
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| City |
Baltimore |
| State/province |
MD |
| ZIP/Postal code |
21205 |
| Country |
USA |
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| Platform ID |
GPL13272 |
| Series (1) |
| GSE33986 |
Retrotransposon Ty1 integration targets specifically positioned asymmetric nucleosomal DNA segments in tRNA hotspots |
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| Relations |
| SRA |
SRX109322 |
| BioSample |
SAMN00760825 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM840085_flowcell62_lanedip.filter.R.clones.sequences.txt.gz |
457.2 Kb |
(ftp)(http) |
TXT |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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