|
| Status |
Public on Jan 13, 2020 |
| Title |
AM16144_Scc1-6HA_CEN3del_no_tension_input |
| Sample type |
SRA |
| |
|
| Source name |
Saccharomyces cerevisiae cell
|
| Organism |
Saccharomyces cerevisiae |
| Characteristics |
chip antibody: Scc1-6HA
target: pMET-CDC20
cell cycle stage: Metaphase
tension/no tension: No tension (Nocodazol treatment)
|
| 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. ChIP was performed using anti-HA antibodies
|
| Growth protocol |
Cells were diluted to OD(600)=0.2 and arrested G1 with alpha factor for 3 hours, then released into a metaphase arrest by depletion of Cdc20 for 2 hours. "No tension" condition included addition of nocodazole and benomyl, "Tension" condition did not.
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
Cells were lysed by beating in MP biomedicals FastPrep-24 machine for 30 seconds three 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 Roche 12CA5 anti-HA 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.
|
| |
|
| Library strategy |
ChIP-Seq |
| Library source |
genomic |
| Library selection |
ChIP |
| Instrument model |
Illumina HiSeq 2000 |
| |
|
| Description |
All samples were prepared using a liquid handling robot (Hamilton) by Bianka Baying (EMBL, Heidelberg, Germany). 10ng of each sample mixed with library preparation were assessed according to manufacturer's recommendation (NEBNext Ultra™ DNA Library Prep Kit for Illumina, (#E7370S/L)). Library preparation was done according to manufacturer. Briefly, the Adapter in the Adapter Ligation step was diluted 1:30 in water. The size selection step was performed with Ampure XP Beads from Beckman Coulter (insert size 200-250bp). The PCR amplification was performed in 15 cycles. Finally NEBNext Multiplex Oligos for Illumina (Index Primer Set 1) (#E7335S/L) were used for the Indexing. The quality and quantity of the libraries was checked using the Agilent BioAnalyzer and the Qubit dsDNA HS Assay kit (Invitrogen).
ChIP-Seq was performed using Illumina HiSeq 2000 Sequencer with Requested Read Length HiSeq50 SE.
|
| Data processing |
Basecalls performed using CASAVA
ChIP-Seq reads were mapped with bwa (aln/samse) to sacCer3 and alternative reference (ref_CEN3del) using parameters (q 15)
Unmapped reads were filtered using samtools
rDNA regions were removed from chrXII/chrMito using bedtools (saturated with reads)
Genome_build: sacCer3 & strain with centromere on chromosome III at ectopic (ref_CEN3del)
Supplementary_files_format_and_content: bigwigs were created using bedtools genomeCoverageBed & wigToBigWig (RPM normalization)
|
| |
|
| Submission date |
Sep 20, 2017 |
| Last update date |
Jan 13, 2020 |
| 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 |
GPL13821 |
| Series (2) |
| GSE104082 |
Scc1 ChIP-Seq in a strain where the centromere on chromosome III has been moved to an ectopic site |
| GSE104135 |
Convergent genes shape budding yeast pericentromeres |
|
| Relations |
| BioSample |
SAMN07679846 |
| SRA |
SRX3202739 |
