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Sample GSM5603477 Query DataSets for GSM5603477
Status Public on Feb 04, 2022
Title IP-Rec8_28720_eco1-aa
Sample type SRA
 
Source name Rec8 eco1-aa IP
Organism Saccharomyces cerevisiae
Characteristics chip antibody: Homemade Rec8 antibody
ploidy: Diploid
cell cycle stage: Meiotic prophase I
background strain: SK1
genotype: RPL13A-2xFKBP12 fpr1-delta tor1-1 ndt80-delta Eco1-FRB-GFP
Treatment protocol Cells carrying ndt80Δ deletion were arrested in meiotic prophase I, fixed in 1% formaldehyde for 2hrs before washing twice in TBS and once in FA lysis buffer with 0.1% SDS.
Growth protocol Cells were grown to saturation in YPDA media before transfer to pre-sporulation media (BYTA) for approx. 16 hours. Cells were then diluted to OD(600) = 1.8 and sporulated for 6 hours in SPO media (0.3% Kac) to arrest in prophase I.
Extracted molecule genomic DNA
Extraction protocol Cells were lysed by beating in MP biomedicals FastPrep-24 machine for 60 seconds four 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 homemade anti-Rec8 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 XP bead 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
Data processing Basecalls performed using CASAVA
Reads were trimmed with cutadapt v3.0 (MAPQ 10, min. length 65)
ChIP-Seq reads were mapped with MiniMap2 v2.10-r763-dirty (-ax sr) to S.cerevisiae SK1
Unmapped reads were filtered using samtools v1.9
rDNA regions were removed from chrXII (saturated with reads) using bedtools v2.27.0
chrMito was excluded with samtools
Genome_build: S.cerevisiae SK1
Supplementary_files_format_and_content: bigwigs were created using bedtools genomeCoverageBed & wigToBigWig (RPM normalization)
 
Submission date Sep 29, 2021
Last update date Feb 04, 2022
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)
GSE185018 Rec8 ChIP-seq for wild type, eco1-aa, wpl1Δ and eco1-aa wpl1Δ strains arrested in meiotic prophase I
GSE185021 Eco1-dependent cohesin acetylation anchors chromatin loops and cohesion to define functional meiotic chromosome domains
Relations
BioSample SAMN21900203
SRA SRX12403699

Supplementary file Size Download File type/resource
GSM5603477_IP-Rec8_28720_eco1-aa_SK1_rpm.bw 35.3 Mb (ftp)(http) BW
SRA Run SelectorHelp
Raw data are available in SRA
Processed data provided as supplementary file

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