|
| Status |
Public on Apr 25, 2019 |
| Title |
AM25226 Rec8-3Ha ChIP-seq WT Input |
| Sample type |
SRA |
| |
|
| Source name |
Meiotic yeast cells
|
| Organism |
Saccharomyces cerevisiae |
| Characteristics |
strain background: SK1
chip antibody: none, input
|
| 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 |
Yeast were grown to saturation in YPDA media before transfer to pre-sporulation media (BYTA). Cells were grown to for approx. 16 hours to OD(600) of approx. 4. Cells were diluted to OD(600) = 1.8 and sporulated for 5 hours in SPO media (0.3% Kac) to arrest in prophase. Cells were released from prophase arrest by addition of 1µM β-estradiol and grown for 75 minutes to arrest in metaphase I.
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
Cells were lysed by beating in MP biomedicals FastPrep-24 machine for 1 minute four times with 10 minute intervals on ice. Cell pellets were washed once in FA lysis buffer/0.1% SDS before sonicating for 30 cycles at 30sec on/off intervals. The supernatant was subject to IP with Sigma M2 anti-Flag 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.
|
| |
|
| Library strategy |
ChIP-Seq |
| Library source |
genomic |
| Library selection |
ChIP |
| Instrument model |
Illumina MiniSeq |
| |
|
| Description |
Reads mapped to SK1 only
|
| Data processing |
Basecalls performed using CASAVA
Reads were trimmed with cutadapt (MAPQ 20, min. length 65)
To obtain reads mapping only to SK1;
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 SK1
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 SK1
Unmapped SK1 reads were filtered using samtools
Unmapped SK1 reads were then mapped to pombe
Unmapped reads were filtered using samtools
chrMito was excluded using bedtools
Genome_build: SK1 (g833-1B) + 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 SK1 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 (SK1)), calibrated SK1 chIP bigwigs were created using bedtools genomeCoverageBed & wigToBigWig
|
| |
|
| Submission date |
Dec 10, 2018 |
| Last update date |
Apr 25, 2019 |
| Contact name |
Daniel Robertson |
| E-mail(s) |
[email protected]
|
| 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 |
GPL22715 |
| Series (2) |
| GSE112217 |
Reductional meiosis I chromosome segregation is established by coordination of key meiotic kinases |
| GSE123546 |
Genome-wide binding of the meiosis specific Rec8 cohesin subunit in metaphase I |
|
| Relations |
| BioSample |
SAMN10564364 |
| SRA |
SRX5123300 |
