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| Status |
Public on Jan 25, 2024 |
| Title |
spo11-delta Mre11-HA6 t5 WCE |
| Sample type |
SRA |
| |
|
| Source name |
meiotic yeast cells
|
| Organisms |
Saccharomyces cerevisiae; Nakaseomyces glabratus |
| Characteristics |
cell type: meiotic yeast cells
genotype: spo11-delta Mre11-HA6
|
| Treatment protocol |
All cells were crosslinked for 15 min in 1 % formaldehyde at room temperature. The formaldehyde was stopped by addition of glycine to 131 mM. For each ChIP, 2x109 S. cer. cells were mixed with 2x108 C. gla. cells which where crosslinked separately. ChIP from meiotic cultures was performed as described in Mendoza et al., Methods Mol Biol. 2009;557:267-83.
|
| Growth protocol |
S. cer. SK1 cells (experimental strain) were induced to sporulate in 2% potassium acetate supplemented with 0.1% (w/v) polypropylene glycol (PPG) to undergo synchronous meiosis at 30°C by the SPS pre-growth method described in Mendoza et al., Methods Mol Biol. 2009;557:267-83. C. gla. CBS138 cells (calibrating strain) were grown in YPD to reach a density of 0.6 at OD660 (Hu et al., Nucleic Acids Res. 2015).
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
Cells were opened by FastPrep-24™ 5G Instrument (MP Biomedicals) followed by sonication with Bioruptor® Pico instrument. After removing the cell debris, the supernatant was used for the ChIP with anti-HA antibody. To reverse crosslink, all the samples were incubated in 65°C overnight. DNA purification was conducted by regular phenol/chloroform/isoamyl alcohol extraction.
Library preparation was performed using NEBNext Ultra II DNA Library Prep Kit according to the manufacturer´s protocol. Amplified library DNA was purified and subjected to 300 bp - 500 bp size selection using Agencourt AMPure XP beads (Beckman Coulter) and quantified by NanoDrop 3300 Fluorospectrometer and Agilent 2100 Bioanalyzer.
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| |
|
| Library strategy |
ChIP-Seq |
| Library source |
genomic |
| Library selection |
ChIP |
| Instrument model |
Illumina HiSeq 2500 |
| |
|
| Data processing |
Unaligned bam files were converted to fastq files with the help of SamToFastq (picard v2.20.5). After removal of adapter sequences with cutadapt (-O 1 -e 0.15 -m 15 –max-n 3), read pairs were aligned to both the ASMv1 and the calibration genome CBS138 with NextGenMap (“global”, allowing up to 4% error), in order to get specific alignments from either species.
Aligned read pairs were "filled up" to cover the whole insert length before calculation of the depths per position ("filled dpp") with a custom Java script. Calibration factors (see supplementary file) were calculated by multiplication of the occupancy ratio (OR), IP.asmv1 x WCE.cbs138 / IP.cbs138 x WCE.asmv1, with the read count normalization factor (to 10M, RP10M).
For calibration the depth counts were multiplied by the sample-specific calibration factors (see also supplemental file) and rounded to three digits.
Assembly: Reference genome version ASM205788v1 (GenBank accession GCA_002057885.1; "ASMv1") for S. cer. SK1; genome version s02-m07-r01 for C. gla. CBS138
Supplementary files format and content: For each ChIP-seq sample, calibrated filled dpp files (chrom, position, depth) are provided.
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| |
|
| Submission date |
Jan 15, 2024 |
| Last update date |
Jan 25, 2024 |
| Contact name |
Franz Klein |
| E-mail(s) |
franz.klein@univie.ac.at
|
| Organization name |
Max Perutz Labs, University of Vienna
|
| Department |
Chromosome Biology
|
| Street address |
Dr. Bohr-Gasse 9
|
| City |
Vienna |
| ZIP/Postal code |
1030 |
| Country |
Austria |
| |
|
| Platform ID |
GPL34086 |
| Series (1) |
| GSE253302 |
Physical interaction with Spo11 mediates the localisation of Mre11 to chromatin in meiosis and promotes its nuclease activity |
|
| Relations |
| BioSample |
SAMN39447976 |
| SRA |
SRX23224027 |
