|
| Status |
Public on Feb 22, 2024 |
| Title |
YLG135, IAA, RPA ChIP, 4h, rep1 |
| Sample type |
SRA |
| |
|
| Source name |
YLG135
|
| Organism |
Saccharomyces cerevisiae |
| Characteristics |
material: Cells
strain: YLG135
genotype: dbf4-3AID
treament: IAA
chip antibody: anti-RFA antibody (Cat# AS07-214, Agrisera)
|
| Treatment protocol |
DSB induction (pGAL-HO system) or synthetic expression of DDK (pGAL-DDK), were achieved by adding 2% galactose in the cultures. Cells were crosslinked at the indicated time points after DSB induction with 1% formaldehyde for 16 minutes at room temperature and the reaction quenched with glycine (final concentration 400 mM). Cells were harvested by centrifugation (3500xg at 4°C), washed with cold PBS and the pellet flash frozen in liquid nitrogen.
|
| Growth protocol |
Cells were grown in YP-media supplemented with 2% raffinose at 30°C. Exponentially growing cells (OD600 0.5-0.7) were synchronized in M phase with 5 μg/ml nocodazole or in G1 phase with 0.5 µg/ml of alpha-factor for 2-3 hours. Cell cycle arrest was assessed via microscopy and DNA content measured by flow cytometry.
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
Cells were mechanically lysed and chromatin was sheared by sonication to fragments of 200-500 bp. 40% of the chromatin extract was used for immunoprecipitation, the purified complexes were treated with proteinase K and crosslinks reversed. DNA was purified by phenol-chloroform extraction and ethanol precipitation.
Strand-specific ChIP-seq libraries were prepared starting from 1 ng of DNA using Accel-NGS® 1S Plus Library Kit, following manufacturer instructions. 12-14 cycles were used for library amplification. Clean-up steps were performed using SPRIselect beads.
|
| |
|
| Library strategy |
ChIP-Seq |
| Library source |
genomic |
| Library selection |
ChIP |
| Instrument model |
Illumina NextSeq 500 |
| |
|
| Data processing |
Sequecing reads were mapped to the Saccharomyces cerevisiae genome using Bowtie2 (v2.4.2) with the parameters --no-discordant --no-mixed --local
SAMtools (v1.9) was used to filter out multiple mapping reads and select reads with alignment quality higher than 30 (MAPQ > 30)
Forward and reverse reads were separated using SAMtools
bedtools (v2.30.0) was used to generate coverage files (bedgraph format)
Assembly: LYZE00000000.1 (HML and HMR loci were removed from the sequence)
Supplementary files format and content: bedgraph files contain reads coverage
|
| |
|
| Submission date |
May 26, 2023 |
| Last update date |
Feb 22, 2024 |
| Contact name |
Martina Peritore |
| E-mail(s) |
peritore@biochem.mpg.de
|
| Phone |
+49 8985783049
|
| Organization name |
MPIB
|
| Lab |
Pfander
|
| Street address |
Am Klopferspitz 18
|
| City |
Planegg-Martinsried |
| State/province |
Bayern |
| ZIP/Postal code |
82152 |
| Country |
Germany |
| |
|
| Platform ID |
GPL19756 |
| Series (1) |
| GSE233549 |
Dbf4-dependent kinase promotes cell-cycle controlled resection of DNA double strand breaks and repair by homologous recombination |
|
| Relations |
| BioSample |
SAMN35439465 |
| SRA |
SRX20524742 |
