|
| Status |
Public on Aug 12, 2015 |
| Title |
Sfc6-Myc ChIP-seq |
| Sample type |
SRA |
| |
|
| Source name |
fission yeast cells, WT, Sfc6-Myc, Myc ChIP
|
| Organism |
Schizosaccharomyces pombe |
| Characteristics |
strain/background: SPK886
genotype/variation: h+N leu1-32 ade6-216 ura4-DS/E Sfc6-13Myc::kanMX
tagged protein: Sfc6-Myc
culture condition: asynchronous cultures
chip antibody: rabbit polyclonal anti-Myc (Novus Biologicals, Cat. NB600-336, Lot. A5)
|
| Growth protocol |
Fission yeast cells (5 x 10^8 cells at 1 x 10^7 cells/ml) were grown at 30°C in YEA medium for Myc ChIP samples. For FLAG ChIP samples, cells were grown at 30°C in EMM medium and further cultured in EMM containing 15 mM thiamine and 0.5 mM auxin (1-naphthaleneacetic acid) for 2 hours (5 x 10^8 cells at 1 x 10^7 cells/ml), followed by pFA fixation. Using an auxin-based degron system (Kanke et al., 2011, BMC Cell Biol vol.12: 8), endogenous Cnd2 proteins were depleted by adding auxin and thiamine into culture medium. Wild-type and mutant Cnd2 proteins tagged with 3FLAG at their C-termini were continuously expressed from plasmids carrying the endogenous cnd2 promoter.
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
Fission yeast cells grown at 30°C in YEA were shifted to 18°C for 5 min before 30 min fixation in 3% paraformaldehyde. Soluble chromatin fractions prepared from fixed cells were sonicated for 30 min by a Bioruptor (Diagenode). Tagged proteins were purified by each antibody and protein G-coupled Dynabeads (Life Technologies).
Libraries were prepared from 5 ng of DNA using the TruSeq DNA sample prep kit (Illumina) for Myc samples or using the NEBNext® ChIP-Seq Library Prep Master Mix Set for Illumina® with the NEBNext® Multiplex Oligos for Illumina® (index Primers Set 1) for FLAG samples, following manufacturer's protocol. Sequencing was performed by an Illumina Genome Analyzer II (Myc) or an Illumina HiSeq 2000 (FLAG).
|
| |
|
| Library strategy |
ChIP-Seq |
| Library source |
genomic |
| Library selection |
ChIP |
| Instrument model |
Illumina Genome Analyzer II |
| |
|
| Data processing |
Basecalls performed using Illumina CASAVA version 8.
ChIP-seq reads were aligned to the S. pombe genome (20090706; download from http://ebi.edu.au/ftp/databases/pombase/pombe/Chromosome_contigs/OLD/20090706/) using Maq version 0.7.1 for MyC samples or aligned to the S. pombe genome (ASM294v1.18; download from ftp://ftp.ensemblgenomes.org/pub/fungi/release-18/fasta/schizosaccharomyces_pombe/dna/) using BWA version 0.6.2 with the option -n 100 for FLAG samples.
Fragment size was estimated by using HOMER version 4.4.
The scores of the peaks were determined by the subtraction of control read density from treatment read density and calculate 200bp window average.
Genome_build: 20090706
Supplementary_files_format_and_content: wig files. Scores represent subtraction of control read density from treatment read density.
|
| |
|
| Submission date |
Aug 08, 2014 |
| Last update date |
May 15, 2019 |
| Contact name |
Hideki Tanizawa |
| E-mail(s) |
hidekit@uoregon.edu
|
| Organization name |
University of Oregon
|
| Department |
Institute of Molecular Biology
|
| Street address |
1370 Franklin Blvd
|
| City |
Eugene |
| State/province |
OR |
| ZIP/Postal code |
97403 |
| Country |
USA |
| |
|
| Platform ID |
GPL9453 |
| Series (1) |
| GSE60273 |
Interaction between TBP and condensing drives the organization and faithful segregation of mitotic chromosomes |
|
| Relations |
| BioSample |
SAMN02980032 |
| SRA |
SRX673287 |
