|
| Status |
Public on Feb 04, 2022 |
| Title |
IP-Smc3_29316_smc3-2r |
| Sample type |
SRA |
| |
|
| Source name |
Smc3 smc3-2R IP
|
| Organism |
Saccharomyces cerevisiae |
| Characteristics |
chip antibody: Homemade rabbit Smc3 antibody
ploidy: Diploid (S.c.) / Haploid (S.p.)
cell cycle stage: Meiotic prophase I
calibration genome: S. pombe cells
background strain: SK1
genotype: smc3::pCLB2-3HA-SMC3 SMC3-K112R,K113R ndt80-delta
|
| Treatment protocol |
Cells were 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.
S. pombe culture at OD600=0.4 was fixed in 1% formaldehyde for 2hrs, washed twice in TBS and once in FA lysis buffer with 0.1% SDS, and aliquoted into equal pellets all equivalent to 100ml of the original culture. Each S. cerevisiae pellet (4 pellets per sample, each obtained from 50ml of meiotic culture at about OD600=2) was mixed with one S. pombe pellet, for a total of 4 S. cerevisiae and 4 S. pombe pellets per sample.
Overall, each sample contains S. cerevisiae and S. pombe cells in an approximate ratio of 2:1. Mixed pellets were lysed by beating.
|
| 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-Smc3 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 only/calibrated to S.pombe
|
| Data processing |
Basecalls performed using CASAVA
Reads were trimmed with cutadapt v3.0 (MAPQ 10, min. length 65)
To obtain reads mapping only to S.cerevisiae SK1;
ChIP-Seq reads were first mapped with MiniMap2 v2.10-r763-dirty (-ax sr) to pombe
Unmapped pombe reads were filtered using samtools v1.9
Unmapped pombe reads were then mapped to SK1
Unmapped reads were filtered using samtools
rDNA regions were removed from chrXII (saturated with reads) using bedtools v2.27.0
chrMito was excluded with samtools
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: S.cerevisiae SK1 + 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 |
Sep 29, 2021 |
| Last update date |
Feb 04, 2022 |
| 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) |
| GSE185020 |
Calibrated Smc3 ChIP-seq for wild type, smc3-2R, wpl1Δ and smc3-2R 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 |
SAMN21900241 |
| SRA |
SRX12403732 |
