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| Status |
Public on Nov 10, 2023 |
| Title |
Rtt101-ChEC_Gal_30sec_rep2 |
| Sample type |
SRA |
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| Source name |
yeast cell
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| Organism |
Saccharomyces cerevisiae |
| Characteristics |
genotype: bar1D::KAN; Flp-nick microscopy (CirO; LEU2-pGAL10-flp-H305L; URA3-FRT-tetOx224(iYFR020W); Rtt101-3xFLAG-Mnase-HPH; MATa
cell type: yeast cell
medium: Galactose-containing media
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| Growth protocol |
Experiments were performed as described in (Gill et al., 2020) with minor modifications. Yeast strains were cultured in YEP medium supplemented with 2% raffinose overnight. Cells were diluted to OD600 = 0.25 in 130 mL of YEP-2% raffinose for 3 h. Then, 50 mL was separated for the induction of flp-H305L expression with 3% galactose for 2 h.
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
For each condition, 50 mL of cultures were harvested at room temperature at 1,500 x g for 30 seconds. Cells were quickly resuspended in 1 mL of Buffer A (15 mM Tris pH 7.5; 80 mM KCl; 0.1 mM EGTA; 0.2 mM spermine; 0.5 mM spermidine; 1 mM PMSF; protease inhibitor cocktail), transferred into 2 mL tubes and pelleted at room temperature at 1,500 x g for 30 seconds. Cells were permeabilized by resuspending in 693 mL of Buffer A supplemented with 7 mL of 10% digitonin and incubated at 30°C with shaking for 5 minutes. An aliquot of 100 mL was separated and kept as a negative control (no MNase digestion). Then, 3.5 mL of 1 M CaCl2 (5 mM final) was added and tubes were immediately placed at 30°C to start MNase cleavage. An aliquot of 100 mL was taken at 30 seconds and immediately mixed with 100mL of Stop Buffer (400 mM NaCl; 20 mM EDTA; 4 mM EGTA; 0.1% SDS). The procedure was repeated for later timepoints. Once all timepoints were collected, cells were treated with 4 mL of 20 mg/mL proteinase K and incubated 30 minutes at 55°C. DNA was then extracted by the addition of 200 mL of phenol:chloroform:isoamyl followed by 5 min centrifugation at maximum speed at room temperature. Then, 150 mL of the aqueous phase was transferred into a new tube, and DNA was precipitated by the addition of 1.5 mL of 20 mg/mL glycogen and 500 mL of 100 % ethanol and incubated at -20°C overnight. Precipitated DNA was centrifuged at maximum speed at 4°C for 10 minutes. DNA pellets were washed with 1 mL of 70% ethanol and air-dried for 5 minutes. Dried pellets were resuspended in 48 mL of 10 mM Tris-HCl pH 8.0 and 2 mL of 10 mg/mL RNase A was added for RNA digestion at 37°C for 30 minutes. For size selection, 100 mL of AmPure XP beads (Beckman Coulter, cat. A63881) was added and the bead:DNA mixture was incubated at room temperature for 5 minutes. Beads were collected by placing tubes on a magnetic rack, and the supernatant was transferred to a new tube containing 55 mL of 10 mM Tris-HCl pH 8.0 and 4 mL of 5 M NaCl. Another DNA extraction and precipitation was performed as described above. DNA pellets were washed and resuspended in 50 mL of ultrapure water. The concentration was determined using a QuBit dsDNA high-sensitivity assay (Invitrogen, cat. Q32854).
NEBnext Ultra DNA library prep kit for Illumina (NEB, cat. E7370L)
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| Library strategy |
OTHER |
| Library source |
genomic |
| Library selection |
other |
| Instrument model |
Illumina NovaSeq 6000 |
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| Description |
Rtt101-ChEC_Gal_30s_0-200bp_mean.bigwig
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| Data processing |
Adapters were removed from the paired-end reads using the Trim Galore! function with default options from the Galaxy server (Afgan et al., 2018; Galaxy, 2022). Trimmed paired-end reads were then aligned to the modified version of the sacCer3 (called sacCer3-Flp-nick and shared as a fasta file) containing an insertion of FRT sites together with a URA3 marker in between the ROG3 and ATG18 coding genes. The bamCoverage function of DeepTools 2.0 (Ramirez et al., 2016) was used to create bigWig density files with the following options: a bin size of 1bp, counts per million (cpm) normalization with the exclusion of the chrM, 20 as a minimum Phred quality score and centering regions with respect to the fragment length limited to 200bp. BigWig files were visualized using Integrative Genomic Viewer (IGV) (Robinson et al., 2011). The average of the two replicates and the differences between the ChEC signal in Galactose and Raffinose-containing media were produced using the bigWigCompare function of DeepTools 2.0.
Assembly: Modified sacCer3 (sacCer3-Flp-nick)
Supplementary files format and content: bigWig files
Library strategy: ChEC-seq
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| Submission date |
Nov 10, 2023 |
| Last update date |
Nov 10, 2023 |
| Contact name |
Julien Soudet |
| E-mail(s) |
julien.soudet@unige.ch
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| Organization name |
Université de Genève
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| Department |
BICEL
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| Street address |
30 quai Ansermet
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| City |
Geneva |
| ZIP/Postal code |
1204 |
| Country |
Switzerland |
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| Platform ID |
GPL27812 |
| Series (1) |
| GSE247492 |
The cullin Rtt101 promotes ubiquitin-dependent DNA-Protein Crosslink repair across the cell cycle |
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| Relations |
| BioSample |
SAMN38201566 |
| SRA |
SRX22483804 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM7890709_Rtt101-ChEC_Gal_30s_0-200bp_rep2.bigwig |
28.6 Mb |
(ftp)(http) |
BIGWIG |
SRA Run Selector |
| Raw data are available in SRA |
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