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| Status |
Public on Jun 09, 2021 |
| Title |
dDSB Spo11-myc18 wt t4 replicate 1 WCE |
| Sample type |
SRA |
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| Source name |
meiotic yeast cells
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| Organisms |
Saccharomyces cerevisiae; Saccharomyces kudriavzevii |
| Characteristics |
strain: SK1 + ZP591
antibody: none
index 1: TCCGCGAA
index 2: AGGCGAAG
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| Growth protocol |
Diploid SK1 strains were induced to undergo meiosis synchronously and were sampled at desired time points in sporulation medium. Diploid SK1 yeast strains from a frozen stock were grown on YPD plates for 2 days at 30°C. Single colonies were inoculated in 5ml YPD and grown over night at 30°C. For synchronous sporulation, the saturated YPD cultures were inoculated in SPS pre-sporulation medium (0.5% Yeast extract, 1% Peptone, 0.17% Yeast nitrogen base (without amino acids and without ammonium sulfate), 1% potassium acetate, 1% ammonium sulfate, 0.05 M potassium biphtalate, pH 5.5) and grown, shaking vigorously, to an OD660 of 1.0 (4x10^7cells/ml). Cells were harvested by centrifugation, washed once and resuspended in sporulation medium (SPM, 2% potassium acetate, 0.1% polypropylene glycol). The meiotic progression of the cells was monitored by DAPI staining and/or western blot analysis.
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
At the desired time points, cells were harvested from 100 ml meiotic cultures and collected by centrifugation (4 min, 2,000 rpm , 4°C), washed once with ice-cold 1xTBS, centrifuged again and pellets were frozen at -80°C. Cells pellets were lysed in 2.4 ml ice-cold lysis buffer (50 mM Hepes-KOH pH=7.5, 150 mM NaCl, 5 mM EDTA, 1% Trition X-100, 0.1% Na-deoxycholate, 1 protease inhibitor tablet (Roche, Complete Protease Inhibitor Cocktail)). For spike-in experiments, 20-30 % of S. kud. meiotic cells were added to S. cer. cells. All steps were carried out at 4°C unless stated. Cells were opened with glass beads 2 x 20 sec, 5.5 m/s in a FastPrep-24™ 5G Instrument (MP Biomedicals). The cell lysates were centrifuged at full speed for 10 min at 4°C and the supernatants were transferred into new Eppendorf tubes. A pre-clearing step was performed by incubating the soluble chromatin fractions with Dynabeads Pan Mouse IgG magnetic beads (Invitrogen) for 1 hour at 4°C. After removing 40 µl of the lysate for the whole cell extract (WCE), chromatin immunoprecipitation was carried out by incubating the lysates with Dynabeads Pan Mouse IgG magnetic beads (Invitrogen) coupled to anti-myc antibody (9E11, mouse monoclonal AB) for 3 hours at 4°C. Thereafter, the beads were washed 2 times with 1 ml lysis buffer, 2 times with 1 ml lysis buffer high salt, 3 times with 1 ml washing buffer (10 mM Tris-HCL pH=8.0, 250 mM LiCl, 0.5% NP-40, 0.5% Na-deoxycholate, 5 mM EDTA) and finally 2 times with 1 ml 10 mM Tris-HCl pH=8.0. Unprotected DNA or partially protected DNA was digested with 3 µl ExonucleaseV (NEB) in NebBuffer 4 (50 mM K-acetate, 20 mM Tris-acetate, 10 mM Mg-acetate and 1 mM DTT at pH=7.9 containing 1 mM ATP) for 30 min at 37°C. In parallel, RNA was removed by incubation with 10 µl RNase (Dnase-free, Roche). The reaction was stopped by the addition of 11 mM EDTA, the beads were washed 3 times with ice-cold 1xTE and incubated at 65°C for 30min in elution buffer (50 mM Tris-HCL pH=8.0, 10 mM EDTA, 1 % SDS). Spo11-protected DNA complexes were digested over night using 12 µl Proteinase K (Sigma) at 50°C. DNA was extracted once with phenol/chloroform/isoamyl alcohol (25:24:1) and precipitated with 0.5 μg/μl glycogen (Roche), 200 mM NaCl and 2.5 volumes of 96 % ethanol. The DNA was pelleted by centrifugation at 4°C for 1 hour, washed once with 1 ml of 70% ethanol and centrifuged again. After air-drying the pellet for 5 min, the DNA was resuspended in 60 µl 10 mM Tris-Cl pH=8.0. The WCE control was digested with RNase and proteinase K, followed by DNA extraction exactly as with the immunoprecipitated samples.
The immunoprecipitated DNA was digested with 3 µl hTdp2 (BPS Bioscience) in 1x hTdp2 Buffer (50 mM Tris-HCl pH7.4, 1 mM MgCl2, 50 mM KCl, 1 mM DTT, 0.1 mg/ml BSA) at 25°C for 30 min. The DNA was extracted with phenol/chloroform/isoamyl alcohol (25:24:1) and precipitated as described above. The hTdp2 digested DNA was end-repaired, A-tailed and adapters were ligated to the DNA ends using the NEBNext Ultra II DNA Library Preparation Kit (NEB) according to the manufacturer´s instructions except that we aimed to select for fragments between 140 bp - 1000 bp length using Agencourt AMPure XP beads (Beckman Coulter). This necessary step likely results in under-representation of the shortest inserts. For amplification, we used either the NEBNext multiplex oligos or the NEBNext dual indexes primer sets. Before library preparation of the WCE control, we fragmented the DNA using NEBNext dsDNA fragmentase (NEB) according to the manufacturer´s protocol. Briefly, we incubated 32µl of the WCE control in 1x fragmentase reaction buffer V2 (50 mM NaCl, 20 mM Tris-HCl, 0.1 mg/ml BSA, 0.15% Triton® X-100, 15 mM MgCl2, pH 7.5@25°C) with 4µl NEBNext dsDNA fragmentase at 37°C for 20 min. The reaction was stopped by the addition of 5 µl of 0.5 M EDTA and the DNA was extracted by phenol/chloroform/isoamyl alcohol (25:24:1) and precipitated as described above. The library preparation of the WCE control was performed the same way as for the immunoprecipitated samples. The concentration and the molarity of the library were determined by NanoDrop™ 3300 Fluorospectrometer and Agilent 2100 Bioanalyzer.
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| Library strategy |
OTHER |
| Library source |
genomic |
| Library selection |
other |
| Instrument model |
Illumina HiSeq 2500 |
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| Data processing |
library strategy: Protec-seq
Unaligned bam files were converted to fastq files with the help of SamToFastq.jar (picard-tools v1.119). Adapter sequences were trimmed by cutadapt (version 1.8.1) with a maximum error rate of 0.15 and minimum clipped read length of 15 nt (options -O 1 -e 0.15 -m 15 --max-n 3). Clipped read pairs were aligned with the help of NextGenMap (version 0.5.5) to both ASMv1 and the calibration genome ZP591 in order to obtain specific S. cer. and S. kud. alignments, as well as read counts for both the ChIP and the WCE samples. Reads aligning to both genomes, typically only 0.2% of the clipped reads, were discarded. In order to map the 5-prime ends accurately and sensitively, global (end-to-end) alignments, a maximum error rate of 4%, and a kmer-size of 9 were applied. In addition, a maximal insert size of 1000nt was set (upper limit of Illumina sequencing machines), and multiple hits were discarded.
Calibration factors (see supplementary file) were calculated by multiplication of the occupancy ratio (OR), IP.asmv1 x WCE.zp591 / IP.zp591 x WCE.asmv1, with the read count normalization factor (to 10M, RP10M), 10M/IP.asmv1. Conversion of alignment data to various data formats representing dDSB fragments or their 5-prime ends were performed with custom Java scripts. dDSB fragment lengths were determined by the distance between start and ends of read pairs (see fd files). Since these fragments include the 2 nt overhangs generated by the Spo11 staggered cuts at both ends, the cleavage axis distances are obtained by subtracting 2 nt from the respective original fragment lengths.
Wiggle files were created with the help of IGVTools v. 2.3.72.
Genome_build: ASM205788v1 (GenBank accession GCA_002057885.1; "ASMv1") for S. cer.; ZP591 (GenBank accession GCA_000167075.2) for S. kud.
Supplementary_files_format_and_content: For each Protec-Seq IP sample, wiggle files of dDSB fragment 5-prime ends are provided as gz-compressed file archives containing one file each for Watson and Crick (numbers preceding the .wig file extension correspond to the RGB color code). Likewise, fragment depth (fd) files containing chromosome, start, length and depth of each dDSB fragment are listed.
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| Submission date |
Mar 29, 2021 |
| Last update date |
Jun 09, 2021 |
| Contact name |
Franz Klein |
| E-mail(s) |
franz.klein@univie.ac.at
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| Organization name |
Max Perutz Labs, University of Vienna
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| Department |
Chromosome Biology
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| Street address |
Dr. Bohr-Gasse 9
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| City |
Vienna |
| ZIP/Postal code |
1030 |
| Country |
Austria |
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| Platform ID |
GPL29940 |
| Series (2) |
| GSE171042 |
Spo11 generates gaps through concerted cuts at sites of topological stress [dDSB] |
| GSE171046 |
Spo11 generates gaps through concerted cuts at sites of topological stress |
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| Relations |
| BioSample |
SAMN18525461 |
| SRA |
SRX10466746 |
| Supplementary data files not provided |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
| Processed data are available on Series record |
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