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| Status |
Public on Jul 27, 2023 |
| Title |
TFIIE-HA, 25 min, ChIP [TF2EHA25] |
| Sample type |
SRA |
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| Source name |
diploid
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| Organism |
Saccharomyces cerevisiae |
| Characteristics |
cell type: diploid
strain: W303
target: TFIIE
tag: HA
promoter: inducible
time: 25 min
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
The parental diploid strain W303 (Ralser et al., 2012) was used to generate all of the competition ChIP strains. For each GTF, one allele was N-terminally tagged with 3xHA and placed under the control of an inducible GAL1 promoter. The other allele was N-terminally tagged with 9xMyc and remained under the control of the endogenous promoter (Longtine et al., 1998). For construction of the GAL1-induced alleles, the plasmid pFA6-His3MX6-PGAL1-3HA (RRID:Addgene_41610, ref. (Longtine et al., 1998)) was used to obtain the HisMX6-PGAL1-3HA cassette by PCR amplification and was integrated into the genome using standard yeast molecular biology techniques. For the GTFs TFIIA, TFIIE, and TFIIF which consist of two subunits, one copy of each subunit was placed under GAL1 control to ensure balanced expression of the competitor isoform. Following integration of the HIS3-GAL1-3HA cassette at one gene subunit, the strain was transformed with the TRP1-GAL1 cassette from pFA6-TRP1-PGAL1 (RRID:Addgene_41606, ref. (Longtine et al., 1998)), placing the second subunit under GAL1 control but without an epitope tag. The 9xMyc tag was integrated into the genome of another isolate of W303 using the integration and Cre-recombinase knockout method and reagents developed by Gauss et al (Gauss et al., 2005). The 9xMyc tag and loxP-flanked KanMX6 marker were PCR amplified from pOM20 and integrated into the yeast genome using standard methods as above. The KanMX6 marker was then then deleted using the GAL-inducible Cre recombinase carried on the plasmid pSH47 (Güldener et al., 1996). The Myc-tagged strains were then transformed with pRS319 (RRID:Addgene_35459, ref. (Sikorski & Hieter, 1989)) to introduce a LEU3 marker for selection. In subsequent steps, diploid strains with HA- or Myc-tagged alleles were sporulated and haploid segregants were mated to yield the competition ChIP (CC) strains with different tags on each of the alleles for the GTF of interest. Proper integration and function of the targeted alleles were confirmed for all strains by PCR, Western blotting using anti-HA or anti-Myc antibodies, and targeted DNA sequencing of the modified loci. Each competition ChIP (CC) strain was inoculated in 100 ml YEP+2% raffinose at 30 °C and incubated overnight. These starter cultures were then used the next day to inoculate 2,250 ml cultures of YEP+2% raffinose at an initial OD600 of 0.05. When an OD of 0.6 was reached, for the 0 minute timepoint 250 ml of the culture was crosslinked by adding 6.75 ml formaldehyde (Thermo Fisher Scientific Cat# F79-500) to achieve a final concentration of 1% for 20 minutes. The reaching was then quenched by adding 15 ml of 2.5M glycine for 5 minutes and the cells were collected by centrifugation. To the rest of the 2,000 ml culture, 142.8 ml of 30% galactose was added to yield a final concentration of 2%. At 10, 20, 25, 30, 40, 60, 90, and 120 minute time points, 250 ml of the culture was collected, crosslinked, and quenched as for the 0 min time point. Cell pellets were washed 3 times with TBS buffer (40 mM Tris-HCl, pH 7.5 plus 300 mM NaCl) and ChIP was performed as described (Viswanathan et al., 2014). The HA and Myc antibodies used for ChIP were the same as those used for western blotting described above. Successful ChIP was confirmed by RT-PCR using primers to detect binding to the URA3 promoter (5’-AAGATGCCCATCACCAAAA-3’ and 5’-AAGAATACCGGTTCCCGATG-3’).
ChIP-seq libraries were prepared following the manufacturer’s instructions using the Illumina TruSeq ChIP library prep kit set A and B (Cat# IP-202-1012 and IP-202-1024). Successful amplification was confirmed by RT-PCR using the URA3 promoter primers. Library quality was assessed using an Agilent Bioanalyzer 2100 and the Agilent-1000 DNA kit (Agilent Cat# 5067-1504), and libraries were quantified using the Qubit dsDNA Quantitation, High Sensitivity kit (Cat# Q32851). A 5nM pool of each library was sequenced on by the UVA Genome Analysis and Technology Core (RRID:SCR_018883) using Illumina NextSeq500 and NextSeq2000 instruments.
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| Library strategy |
ChIP-Seq |
| Library source |
genomic |
| Library selection |
ChIP |
| Instrument model |
NextSeq 2000 |
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| Description |
HATF2E25
ha_tfiie.txt
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| Data processing |
Reads were mapped to the sacSer3 reference genome using Bowtie2 (v2.2.6) with default settings.
Peaks of enrichment were identified using MACS2 (v2.1.0.20151222) applied to each of several early time point Myc datasets and using an input dataset as control and --nomodel --extsize 147.
Count tables were then generated by associating reads with the peak intervals using bedtools multicov.
Read counts were normalized in a three-step process. First, read counts in each peak and for each time point were normalized to the overall read depth. Next, read counts for the HA samples were normalized to the average relative levels of the factor of interest using the average values obtained from three independent western blots. Lastly, the normalized HA read count matrix was divided by the normalized Myc count matrix to yield the ratio count tables for mathematical modeling.
Assembly: sacCer3
Supplementary files format and content: tab delimited text files containing raw read counts
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| Submission date |
Jun 14, 2023 |
| Last update date |
Jul 27, 2023 |
| Contact name |
David T. Auble |
| E-mail(s) |
[email protected]
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| Organization name |
University of Virginia
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| Department |
Biochemistry and Molecular Genetics
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| Street address |
1307 Lane Rd
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| City |
Charlottesville |
| State/province |
VA |
| ZIP/Postal code |
22903 |
| Country |
USA |
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| Platform ID |
GPL31112 |
| Series (2) |
| GSE235000 |
Genome-scale chromatin interaction dynamic measurements for key components of the RNA Pol II general transcription machinery [ChIP-seq] |
| GSE235002 |
Genome-scale chromatin interaction dynamic measurements for key components of the RNA Pol II general transcription machinery |
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| Relations |
| BioSample |
SAMN35737855 |
| SRA |
SRX20682351 |
| Supplementary data files not provided |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data are available on Series record |
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