 |
 |
GEO help: Mouse over screen elements for information. |
|
| Status |
Public on Nov 10, 2023 |
| Title |
Rtt101-HA_ChIP_G1_rep1 |
| Sample type |
SRA |
| |
|
| Source name |
yeast cell
|
| Organism |
Saccharomyces cerevisiae |
| Characteristics |
genotype: Rtt101-3HA-HIS3; Pol2-5Flag-KAN; bar1D::URA3; MATa
cell type: yeast cell
treatment: G1-phase arrested cells
chip antibody: anti-HA (BioLegend 901502)
|
| Growth protocol |
Yeast cells were grown in YEPD until exponential phase, and synchronized in G1 phase by addition of α-factor (ref) for 2 hours. After two washes, cells were released into the cell cycle in fresh YEPD medium without drugs, or supplemented with 200 mM of HU.
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
Cells were crosslinked with 1 % formaldehyde for 15 min at room temperature, quenched with 250 mM glycine incubated for 5 min, followed by ice-cooling for at least 10min. Before freezing, pellets were washed twice with cold 1X Phosphate Buffer Saline (PBS) and transferred into 2 mL screw-cap tubes. All subsequent steps were performed at 4°C or on ice, if not mentioned otherwise. Frozen pellets were resuspended in 1 mL cold FA lysis buffer (50 mM HEPEs-KOH pH 7.5; 140 mM NaCl; 1 mM EDTA; 1 % Triton X-100; 0.1 % sodium deoxycholate; protease inhibitor cocktail) and lysed by MagNA Lyzer (6000 rpm; 5 cycles of 30 secs; 1 min on ice between runs). Extracts were recovered in a new tube and centrifuged at 13,000 x rpm for 30min. After resuspension with 1 mL of fresh FA lysis buffer, extracts were sonicated via Bioruptor Twin (Diagenode) at 4°C for 20 cycles of 30 secs. After centrifugation at 13,000 x rpm for 15 min, supernatant was transferred in a new tube and the amount of protein was determine by Bio-Rad protein assay (cat. 500-0006). Each immunoprecipitation was carried out with 1 mg of protein (1/10 retained as the input) incubated overnight at 4°C under rotation, with 1 uL of anti-HA antibody. Protein G sepharose beads were then added for 3 hours, at 4°C under rotation. After incubation, beads were washed 1x with 500 µL cold FA lysis buffer, 2x with 500 µL cold FA-500 buffer (FA lysis with 500 mM NaCl), 2x with 500 µL Buffer 3 (10 mM Tris-HCl pH 8; 1 mM EDTA; 250 mM LiCl, 1 % IGEPAL; 1 % sodium deoxycholate) and 1x with 500 µL 1X TE (50 mM Tris-HCl pH 7.5; 10 mM EDTA). Elution was then performed twice with 100 µL elution buffer (50 mM Tris-HCl pH 7.5; 1 % SDS; 10 mM EDTA) incubated at 65°C for 8 minutes. Eluted sample and input were treated with 0.75 mg/mL Proteinase K and incubated at 42°C for 2 hours, followed by de-crosslinking overnight at 65°C. Both IP and input were purified with the MinElute PCR Purification kit (Qiagen, cat. 28006), following manufacturer’s recommendations and eluted from the column twice with 30 µL of elution buffer from the kit.
NEBnext Ultra DNA library prep kit for Illumina (NEB, cat. E7370L)
|
| |
|
| Library strategy |
ChIP-Seq |
| Library source |
genomic |
| Library selection |
ChIP |
| Instrument model |
Illumina NovaSeq 6000 |
| |
|
| Data processing |
After adapters removal using Trim Galore!, reads were aligned to the sacCer3 genome via Bowtie2 (Langmead and Salzberg, 2012) through the Galaxy server. BigWig density files were then produced using the bamCoverage function with the following options: a bin size of 250 smoothed by a rolling window of 1000bp, cpm normalization with the exclusion of the chrM, centering fragments after their artificial extension to 250bp and a minimal Phred quality score of 20. Differential densities between time points in S-phase and G1-phase were processed through the bigwigCompare function. Metagene plots were made using the computeMatrix and plotProfile commands. The list of ARS Consensus Sequences (ACSs) was retrieved from (Soudet et al., 2018) and their orientation was taken into account. The 40 early ARSs were defined through a k-means clustering based on the DNA Pol 2 signal 10kb around the ACSs. The median Z-score was finally calculated on the 30kb-window around ACSs.
Assembly: sacCer3
Supplementary files format and content: bigWig files
|
| |
|
| Submission date |
Nov 10, 2023 |
| Last update date |
Nov 10, 2023 |
| Contact name |
Julien Soudet |
| E-mail(s) |
julien.soudet@unige.ch
|
| Organization name |
Université de Genève
|
| Department |
BICEL
|
| Street address |
30 quai Ansermet
|
| City |
Geneva |
| ZIP/Postal code |
1204 |
| Country |
Switzerland |
| |
|
| Platform ID |
GPL27812 |
| Series (1) |
| GSE247492 |
The cullin Rtt101 promotes ubiquitin-dependent DNA-Protein Crosslink repair across the cell cycle |
|
| Relations |
| BioSample |
SAMN38201559 |
| SRA |
SRX22483811 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM7890716_Rtt101_G1_rep1_250-1000.bigwig |
315.4 Kb |
(ftp)(http) |
BIGWIG |
SRA Run Selector |
| Raw data are available in SRA |
|
|
|
|
 |
