|
| Status |
Public on Nov 11, 2016 |
| Title |
Hsf1_ChIP |
| Sample type |
SRA |
| |
|
| Source name |
cell culture
|
| Organism |
Saccharomyces cerevisiae |
| Characteristics |
strain: BY4742
genotype: BY4742 tor1-1; fpr1del; RPL13A-FKBP12-NAT; HSF1-FRB-yEGFP-hphMX4
mating type: MATalpha
mnase concentration: none
depletion time: 0min
|
| Treatment protocol |
For the Mnase-seq material: Hsf1 was depleted from the nucleus by adding rapamycin, dissolved in DMSO, to a final concentration of 7.5 µM. For the “t=0” samples no rapamycin, but the same volume of DMSO was added. Harvesting was done 30 minutes after addition of rapamycin.
|
| Growth protocol |
Strains were streaked from -80oC stocks onto appropriate selection plates and grown for 3 days. Liquid cultures were inoculated with independent colonies and grown overnight (ON) in Synthetic Complete (SC) medium: 2 gr/L Drop out mix Complete and 6.71 gr/L Yeast Nitrogen Base without AA, Carbohydrate & w/AS (YNB) from US Biologicals (Swampscott, USA) with 2% D-glucose. The following day O/N cultures were used to inoculate 250 ml (ChIP-seq) or 70 ml (Mnase-seq) cultures of SC media at OD595=0.35 (ChIP-seq) or OD595=0.15 (Mnase-seq) and were grown for two doublings. All growth was done at 30oC.
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
ChIP was caried out as previously described (van Bakel et al., NAR 2008), Chromatin for Mnase digestion was prepared essentialy as described (Kent and Mellor, 1995)
Sequencing libraries were created from the purified DNA using the NextflexTM rapid DNA-Seq kit (Bioo Scientific) using a modified protocol.
|
| |
|
| Library strategy |
ChIP-Seq |
| Library source |
genomic |
| Library selection |
ChIP |
| Instrument model |
AB 5500xl-W Genetic Analysis System |
| |
|
| Description |
ChIP sequencing of Hsf1-FRB-GFP (without depleting Hsf1 from the nucleus)
The anchor away system (Haruki et al., 2008) was recreated in the Saccharomyces cerevisiae S288C/BY4742 strain. To achieve this FPR1 was deleted, a tor1-1 mutation was introduced to desensitize the strain to rapamycin and RPL13A was tagged with 2xFKBP12 as in the original anchor away system (Haruki et al., 2008). This strain was the parental strain used to create the specific anchor away strains. This strain is referred to as wildtype (WT) throughout the study. In order to create the specific anchor away strains, Hsf1 was tagged with an FRB-yEGFP tag.
|
| Data processing |
The Chip-seq reads were aligned using bwa (Li and Durbin, 2009; version 0.5.9-r16 aln) with the settings ‘-c –l 25 –k 2 –n 10’The Mnase-seq reads were mapped using bowtie2 (Langmead and Salzberg, 2012; version 2.2.5) with the settings ‘ --no-discordant --no-contain --maxins 1980 --trim5 5 --trim3 15 --end-to-end --sensitive’
Only read pairs with an insert size between 95-225 bp were used for subsequent analyses. To compare the occupancy of all samples, their numbers were scaled to 10 million mapped paired-end reads per sample using genomecov from the bedtools2 suite version 2.250 (Quinlan and Hall, 2010).
The nucleosome dyads (midpoints of the mate pairs) were determined and smoothed using a 31 bp running average using https://github.com/plijnzaad/phtools/blob/master/ngs/center+smooth.pl and converted to BigWig format using genomecov from the bedtools2 suite version 2.250 (Quinlan and Hall, 2010) and bedGraphToBigWig (Kent et al., 2010)
Genome_build: Saccharomyces cerevisiae, assembly R64 (aka sacCer3)
Supplementary_files_format_and_content: Processed data files represent the Hsf1 binding peaks, and nucleosome dyad positions, respectively
|
| |
|
| Submission date |
May 23, 2016 |
| Last update date |
May 15, 2019 |
| Contact name |
Marian Groot Koerkamp |
| Organization name |
Princess Maxima Center for Pediatric Oncology
|
| Department |
Research
|
| Lab |
Drostlab
|
| Street address |
Heidelberglaan 25
|
| City |
Utrecht |
| State/province |
Utrecht |
| ZIP/Postal code |
3584 CS |
| Country |
Netherlands |
| |
|
| Platform ID |
GPL21944 |
| Series (2) |
| GSE81481 |
HSF1 and MOT1-expression and binding: Molecular mechanisms that distinguish TFIID housekeeping from regulatable SAGA promoters |
| GSE81787 |
HSF1 ChIP-seq: Molecular mechanisms that distinguish TFIID housekeeping from regulatable SAGA promoters |
|
| Relations |
| BioSample |
SAMN04966256 |
| SRA |
SRX1756249 |
