|
| Status |
Public on Feb 04, 2022 |
| Title |
IP-Rec8_29750_wpl1 |
| Sample type |
SRA |
| |
|
| Source name |
Rec8 wpl1Δ 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 wpl1-delta
|
| 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 |
SAMN21900202 |
| SRA |
SRX12403700 |
