|
| Status |
Public on Jan 13, 2020 |
| Title |
Scc1-6HA WT Tension IP |
| Sample type |
SRA |
| |
|
| Source name |
S. cerevisiae/S. pombe cells
|
| Organisms |
Schizosaccharomyces pombe; Saccharomyces cerevisiae |
| Characteristics |
chip antibody: Mouse monoclonal 12CA5 anti-HA (Roche) against Scc1-6HA/Rad21-6HA
target: pMET-CDC20
cell cycle stage: Metaphase
treatment: Tension
genotype: WT
|
| Treatment protocol |
Cells were fixed in 1% formaldehyde for 30 minutes before washing twice in TBS and once in FA lysis buffer with 0.1% SDS.
|
| Growth protocol |
Cells were diluted to OD(600)=0.2 and arrested G1 with alpha factor for 3 hours, then released into a metaphase arrest by depletion of Cdc20 for 2 hours. "No tension" condition included addition of nocodazole and benomyl, "Tension" condition did not.
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
Cells were lysed by beating in MP biomedicals FastPrep-24 machine for 30 seconds three times with 10 minute intervals on ice. Cell pellets were washed once in FA lysis buffer/0.1% SDS before sonicating in a Bioruptor (Diagenode) for two times 30 cycles at 30sec on/off intervals at "High" setting. The supernatant was subject to IP with Roche 12CA5 anti-HA antibody conjugated to Dynabeads over night. Dynabeads were washed and bound protein eluted using TES buffer. Samples were decrosslinked with Proteinase K and purified using Promega Wizard kit.
DNA was blunted using NEB Quick Blunting kit followed by Ampure purification of fragments over 100bp. dA tails were added using Klenow (exo-) and NextFlex adaptors ligated to each sample. This was followed by two Ampure purifications of fragments over 100bp and then fragments over 150-200bp. Libraries were amplified using NextFlex primers and Phusion DNA polymerase. Lastly, double-sided Ampure purification was carried out to obtain fragments between 100-300bp in size.
ChIP-seq, barcoded
|
| |
|
| Library strategy |
ChIP-Seq |
| Library source |
genomic |
| Library selection |
ChIP |
| Instrument model |
Illumina MiniSeq |
| |
|
| Description |
1105_T_IP_S3
Reads mapped to sacCer3 only/calibrated to S.pombe
|
| Data processing |
Basecalls performed using CASAVA
Reads were trimmed with cutadapt (MAPQ 10, min. length 65)
To obtain reads mapping only to sacCer3;
ChIP-Seq reads were first mapped with MiniMap2 (-ax sr) to pombe
Unmapped pombe reads were filtered using samtools
Unmapped pombe reads were then mapped to sacCer3
Unmapped reads were filtered using samtools
rDNA regions were removed from chrXII (saturated with reads)
chrMito was excluded using bedtools
To obtain reads mapping only to Pombe;
Reads were also mapped to sacCer3
Unmapped sacCer3 reads were filtered using samtools
Unmapped sacCer3 reads were then mapped to pombe
Unmapped reads were filtered using samtools
chrMito was excluded using bedtools
Genome_build: sacCer3 + s.pombe ASM294v2.22
Supplementary_files_format_and_content: bigwigs were created using bedtools genomeCoverageBed & wigToBigWig (RPM normalization), To obtain calibrated bigWigs; samtools flagstat was used to count reads mapping to sacCer3 and pombe only for each sample the occupancy ratio (OR) value was calculated as Wc*IPx/Wx*IPc (W=Input;IP=chIP;c=calibration genome (pombe);x=experimental genome (sacCer3)), calibrated sacCer3 chIP bigwigs were created using bedtools genomeCoverageBed & wigToBigWig
|
| |
|
| Submission date |
Mar 13, 2019 |
| Last update date |
Jan 13, 2020 |
| Contact name |
Daniel Robertson |
| E-mail(s) |
daniel.robertson@ed.ac.uk
|
| Organization name |
University of Edinburgh
|
| Department |
Discovery Research Platform for Hidden Cell Biology
|
| Lab |
Bioinformatics Core
|
| Street address |
2.28 Michael Swann Building, Kings Buildings, Mayfield Road
|
| City |
Edinburgh |
| State/province |
Midlothian |
| ZIP/Postal code |
EH9 3JR |
| Country |
United Kingdom |
| |
|
| Platform ID |
GPL26296 |
| Series (2) |
| GSE104135 |
Convergent genes shape budding yeast pericentromeres |
| GSE128238 |
Calibrated Scc1 ChIP-seq for wild type and chl4Δ strains arrested in metaphase with and without tension |
|
| Relations |
| BioSample |
SAMN11115946 |
| SRA |
SRX5513544 |
