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Sample GSM3946396 Query DataSets for GSM3946396
Status Public on Jul 18, 2019
Title NPAS2_bHLH_CBF1Zip
Sample type SRA
Source name BY4741
Organism Saccharomyces cerevisiae
Characteristics strain: BY4741
genotype: sir4D cbf1D
transcription factor: NPAS2bHLH with Cbf1p ZIP
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).
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.
Library strategy OTHER
Library source genomic
Library selection other
Instrument model Illumina MiniSeq
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
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
Submission date Jul 17, 2019
Last update date Jul 18, 2019
Contact name Christian Shively
Organization name Washington University in St Louis
Department Genetics
Lab Mitra
Street address 4523 Clayton Avenue
City Saint Louis
State/province MO
ZIP/Postal code 63108
Country USA
Platform ID GPL22715
Series (1)
GSE134427 Homotypic cooperativity and collective binding are determinants of bHLH specificity and function
SRA SRX6457367
BioSample SAMN12306985

Supplementary file Size Download File type/resource
GSM3946396_NPAS2_DBD_COIL_ACTGT_GTAGTGCC.gnashy.txt.gz 15.2 Kb (ftp)(http) TXT
GSM3946396_intergenic_regions_NPAS2_DBD_COIL.gnashy.txt.gz 173.8 Kb (ftp)(http) TXT
SRA Run SelectorHelp
Raw data are available in SRA
Processed data provided as supplementary file

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