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| Status |
Public on Jul 18, 2019 |
| Title |
GCR2 |
| Sample type |
SRA |
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| Source name |
BY4741
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| Organism |
Saccharomyces cerevisiae |
| Characteristics |
strain: BY4741
genotype: sir4D
transcription factor: Gcr2p
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| Growth protocol |
The indicated yeast strain was co-transformed with a plasmid carrying the indicated transcription factor fused at the C-terminus to Sir4p and a plasmid carrying a barcoded HIS3-carrying Ty5 retrotransposon under the control of the GAL1/10 promoter. A single clone was then incubated for 3 days on selective media with galactose as the carbon source. Plates were then sequentially replica-plated to YPD (2 days), followed by SC-HIS with 5'FOA agar plates (3 days).
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
Genomic DNA was extracted from all cells using standard glass bead-beating, phenol chloroform isoamyl alcohol purification, and NH4Ac ethanol protocol.
Genomc DNA was digested in three separate restriction enzyme digests (HinPI1, HpaII, and Taq-alphaI), self-ligated with T4 DNA ligase, and an inverse PCR reaction was performed to amplify genomic DNA downstream of each inserted Ty5 retrotransposon.
"Calling Card" Assay to identify in vivo genomic targets of DNA-binding factors
"Calling Card" Assay performed as previously described: 1. "Calling Cards enable multiplexed identification of the genomic targets of DNA-binding proteins." Wang H, Mayhew D, Chen X, Johnston M, Mitra RD. Genome Research, 2011, May 21(5):748-55. PMID 21471402. 2. "Calling Card Analysis in Budding Yeast." Mayhew D, Mitra RD. Cold Spring Harbor Protocols, 2016, Feb 1, 2016(2). PMID 26832687.
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| Library strategy |
OTHER |
| Library source |
genomic |
| Library selection |
other |
| Instrument model |
Illumina MiniSeq |
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| Description |
"Calling Card" Assay for in vivo Transcription Factor Binding: a retrotransposon is directed into the genome at loci bound by a given transcription factor
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| Data processing |
Library strategy: Calling Card
Paired-End sequence reads were filtered for presence of Read1 initial 5bp barcode followed by unique 17-mer sequence (AATTCACTACGTCAACA) and Read2 initial 8bp barcode.
80bp of Read1 insert sequence was mapped to the 2008 S. cerevisiae reference genome (R-61-1-1) sequence using NovoAlign with default parameter settings. (Insertion position and number of reads mapping to each insertion is given in gnashy file.)
Number of insertions into each S. cerevisiae intergenic region was normalized to a total of 100,000 retrotransposon insertions per experiment and p-value was generated using poisson statistics. (Significantly bound intergenic regions file)
Genome_build: 2008 S. cerevisiae reference genome (R-61-1-1)
Supplementary_files_format_and_content: .gnashy - a table indicating genomic position (chromosome, chromosome coordinate) of the retrotransposon insertion and number of reads aligning to that position
Supplementary_files_format_and_content: .gnashy.txt - Significantly bound intergenic regions file giving S. cerevisiae intergenic region, flanking ORFs (standard and systematic names), number of detected retrotransposon "calling card" insertions, normalized number of insertions (TPH), hyper-geometric p-value
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| Submission date |
Jul 17, 2019 |
| Last update date |
Jul 18, 2019 |
| Contact name |
Christian Shively |
| E-mail(s) |
cshively@wustl.edu
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| Organization name |
Washington University in St Louis
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| Department |
Genetics
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| Lab |
Mitra
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| Street address |
4523 Clayton Avenue
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| City |
Saint Louis |
| State/province |
MO |
| ZIP/Postal code |
63108 |
| Country |
USA |
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| Platform ID |
GPL22715 |
| Series (1) |
| GSE134427 |
Homotypic cooperativity and collective binding are determinants of bHLH specificity and function |
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| Relations |
| SRA |
SRX6457364 |
| BioSample |
SAMN12306988 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM3946393_GCR2_Shively2019_All.gnashy.txt.gz |
134.1 Kb |
(ftp)(http) |
TXT |
| GSM3946393_intergenic_regions_GCR2_Shively2019_All.gnashy.txt.gz |
218.0 Kb |
(ftp)(http) |
TXT |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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