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| Status |
Public on Aug 02, 2017 |
| Title |
Taf11_YPD_HS_3IAA_1 |
| Sample type |
SRA |
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| Source name |
Flag IP - Rpb3-3XFlag / Taf11-3V5-IAA7 - YPD 30 deg with 37 deg heat shock 30 min, + 500 uM 3-IAA 30 min
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| Organism |
Saccharomyces cerevisiae |
| Characteristics |
strain: SHY1042
antibody: Anti-FLAG M2 Magnetic Beads
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| Treatment protocol |
Cells were then induced for protein degradation by the addition of 10 ml 200X 3-IAA (500 μM final working concentration) dissolved in DMSO, or 10 ml DMSO for the no 3-IAA controls, for 30 minutes at 30 deg.
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| Growth protocol |
For each yeast strain and condition, two liters of S. cerevisiae were grown at 30 deg in YPD (+ 100 μl Antifoam 204) to an absorbance of ~0.7-1.0.
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
Cells were rapidly harvested by vacuum filtration through a 90 mm diameter 0.45 micron nitrocellulose filter, followed by a 100 ml wash with the cell culture media. The cell paste was scraped off with a pre-chilled spatula and plunged into liquid nitrogen to flash freeze cells. For spike-in normalization, an 8 liter batch of S. pombe was grown in YE (+ Antifoam 204) to a similar absorbance and harvested as described above. 2.5 g S. cerevisiae were combined with 0.25 g S. pombe in a mortar pre-chilled on dry ice and liquid nitrogen. Frozen cells were lysed with a 4.5” OD mortar and large pestle. Cells were ground for 1 min, liquid nitrogen was then added to the cells and ground for 1 min. The grinding and addition of liquid nitrogen was repeated for a total of 6 minutes. The frozen powdered ground cells were transferred to a cold conical tube and stored at -80 deg.
DNA isolated from IPs was prepared for sequencing as previously described (Skene and Henikoff, eLIFE 2017), except for the following changes. End Repair and A-tailing was performed in a 20 ml volume containing up to 50 ng DNA and the following final concentrations: 1X T4 DNA ligase buffer (NEB #B0202S), 0.5 mM each dNTP, 0.25 mM ATP, 2.5% PEG 4000, 2.5 U T4 PNK (NEB #M0201S), 0.05 U T4 DNA polymerase (Invitrogen #18005025), and 0.05 U Taq DNA polymerase and reactions were incubated at 12 deg 15 min, 37 deg 15 min, 72 deg 20 min, then put on ice and immediately used in adapter ligation reactions. Adapter Ligation was performed in a 50 ml volume containing 100:1 adapter:insert molar ratios, 1X Rapid DNA ligase buffer (Enzymatics #B101L) and 3000 U DNA ligase (Enzymatics #L6030-HC-L). A single post-ligation cleanup was performed using 0.4X vol AMPure XP reagent (Beckman Coulter #A63881) and DNA eluted in 40 ul 0.1X TE. Library Enrichment was performed in a 25 μl volume containing 16.25 μl DNA from previous step and the following final concentrations: 1X KAPA buffer, 0.3 mM each dNTP, 0.5 mM each P5 and P7 PCR primer, and 0.5 U KAPA HS HIFI polymerase (#KK2502). DNA was amplified with the following program: 98 deg 45 sec, [98 deg 15 sec, ramp to 60 deg @ 1 deg/sec, 60 deg 20 sec, ramp to 98 deg @ 1 deg/sec] 8-10X, 72 deg 1 min. A post-PCR cleanup was performed using 1.4X vol AMPure XP reagent and DNA was eluted into 40 ml 0.1X TE. Libraries were sequenced on the Illumina HiSeq2500 platform using 25 bp paired-ends at the Fred Hutchinson Cancer Research Center Genomics Shared Resources facility.
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| Library strategy |
ChIP-Seq |
| Library source |
genomic |
| Library selection |
ChIP |
| Instrument model |
Illumina HiSeq 2500 |
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| Description |
Flag Immunoprecipitation
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| Data processing |
We used Bowtie2 to map the paired-end 25bp reads to Saccer3 version of yeast genomic sequence obtained from UCSC.
We used Bowtie2 to map the paired-end 25bp reads to ASM294v2.20 version of pombe genomic sequence obtained from pombase.
We counted the properly paired reads from the S. pombe alignments.
Genome_build: SacCer3
Genome_build: PomBase ASM294v2.20
Supplementary_files_format_and_content: We generated wig files from fragments of size 25-140 bp aligned to S. cerevisiae genome with a scaling factor of (10000/number of fragments mapped to S. pombe) to create spike-normalized coverage file (Supplementary file .wig)
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| Submission date |
Mar 27, 2017 |
| Last update date |
May 15, 2019 |
| Contact name |
Srinivas Ramachandran |
| E-mail(s) |
srinivas.ramachandran@cuanschutz.edu
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| Organization name |
University of Colorado School of Medicine
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| Department |
Biochemistry and Molecular Genetics
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| Street address |
Mail Stop: 8101, 12801 East 17th Ave. L-18-9102
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| City |
Aurora |
| State/province |
CO |
| ZIP/Postal code |
80045 |
| Country |
USA |
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| Platform ID |
GPL17342 |
| Series (1) |
| GSE97081 |
Transcription of nearly all yeast RNA Polymerase II-transcribed genes is dependent on transcription factor TFIID |
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| Relations |
| BioSample |
SAMN06645378 |
| SRA |
SRX2675876 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM2551241_106_140.wig.gz |
41.5 Mb |
(ftp)(http) |
WIG |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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