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Status |
Public on Apr 25, 2019 |
Title |
AM22605 Sgo1-6Ha ChIP-seq WT IP |
Sample type |
SRA |
|
|
Source name |
Meiotic yeast cells
|
Organism |
Saccharomyces cerevisiae |
Characteristics |
strain background: SK1 genotype/variation: wild type chip antibody: Mouse monoclonal 12CA5 anti-HA (Roche)
|
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.
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|
|
Library strategy |
ChIP-Seq |
Library source |
genomic |
Library selection |
ChIP |
Instrument model |
Illumina MiniSeq |
|
|
Description |
Reads mapped to SK1 only/calibrated to s.pombe
|
Data processing |
Basecalls performed using CASAVA Reads were trimmed with cutadapt (MAPQ 10, min. length 30) To obtain reads mapping only to SK1; ChIP-Seq reads were first mapped with bwa (aln/sampe) to pombe Unmapped pombe reads were filtered using samtools Unmapped pombe reads were then mapped to SK1 (bwa mem) 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
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|
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Submission date |
Mar 21, 2018 |
Last update date |
Apr 25, 2019 |
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 |
GPL22715 |
Series (2) |
GSE112167 |
Genome-wide binding of the cohesin protector Sgo1 in the presence and absence of the meiosis I-specific Spo13 protein |
GSE112217 |
Reductional meiosis I chromosome segregation is established by coordination of key meiotic kinases |
|
Relations |
BioSample |
SAMN08767396 |
SRA |
SRX3828984 |