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Sample GSM3059082 Query DataSets for GSM3059082
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.
 
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
 
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

Supplementary file Size Download File type/resource
GSM3059082_Sgo1-6HA_WT_22605-IP_S2.calibrated.bw 18.4 Mb (ftp)(http) BW
GSM3059082_Sgo1-6HA_WT_22605-IP_S2.rpm.bw 18.4 Mb (ftp)(http) BW
SRA Run SelectorHelp
Raw data are available in SRA
Processed data provided as supplementary file

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