 |
 |
GEO help: Mouse over screen elements for information. |
|
| Status |
Public on Apr 02, 2015 |
| Title |
3b_exp |
| Sample type |
SRA |
| |
|
| Source name |
yeast cell culture
|
| Organism |
Saccharomyces cerevisiae |
| Characteristics |
growth phase: exponential growth phase
strain: W303
genotype: wild-type
|
| Treatment protocol |
N/A
|
| Growth protocol |
All the yeast strains were grown at 30 C in SC + Galactose 2% without uracil (Sunrise Science, cat 1652 and 1485-100) overnight. The next morning, cells were diluted to 0.3 OD600/ml and grown to mid-log phase (0.8-1 OD600/ml) or to stationary phase (5-6 OD600/ml or for 24-30 h).
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
5 OD600 of yeast cells were disrupted by vortexing for 6 minutes in a Disruptor Genie (Scientific Industries, Inc.) in the presence of an equal volume of breaking buffer, acid-washed glass beads and phenol:chloroform (1:1). After the addition of TE buffer, aqueous phase is transferred into a new tube and precipitated with ethanol. The nucleic acid pellet is resuspended in TE buffer and treated for 1 hour at 37°C with RNase A, followed by incubation for 1 hour with 2 mg/ml Proteinase K in the presence of 1% SDS at 60°C. The resulting solution is treated twice with phenol:chloroform (1:1), once with chloroform and ethanol precipitated. The DNA pellet is resuspended in EB buffer (Qiagen).
Between 500-1000 ng of extracted yeast DNA are added to 2 ng of λ unmethylated DNA (Promega, D1521) and the mixture is sonicated with a Covaris S-2 to obtain fragments in the 200-300 bp range [Total Time: 6 minutes; Duty Cycle: 10%; Intensity: 5; Cycles/Burst: 200; Mode: Frequency Sweeping]. The reagents used in the library preparation are from the Illumina TruSeq DNA Sample Prep kit v2. End-Repair, purification and dA-tailing steps are performed according to manufacturer’s instructions. Ligation is performed according to the protocol except that 1 μl of Illumina TruSeq Adapters is used in the final reaction. The ligation reaction is purified using 1.2 volumes of AMPure XP beads (Beckman Coulter Inc.) and DNA fragments with a 170-350 bp range are enriched using 0.7 and 0.3 volumes of AMPure XP beads in the first and second size-selection step, respectively. Samples are treated with bisulfite (EpiTect kit, QIAGEN) according to manufacturer’s protocol, except that two consecutive rounds of conversion are performed, for a total of 10 h of incubation. Half of the converted DNA is amplified using MyTaq Mix (Bioline) and Illumina TruSeq PCR Primer Cocktail according to the following protocol: initial denaturation at 98°C for 30 seconds; 12 cycles of 98°C for 15 seconds, 60°C for 30 seconds, 72°C for 30 seconds; final extension at 72°C for 5 minutes. The final product is purified using AMPure XP beads before being submitted for sequencing.
|
| |
|
| Library strategy |
Bisulfite-Seq |
| Library source |
genomic |
| Library selection |
RANDOM |
| Instrument model |
Illumina HiSeq 2000 |
| |
|
| Data processing |
Reads from bisulfite-treated yeast genomic DNA were aligned using BS-Seeker2 v2.0.3 (Guo et al., 2013) against the sacCer3 and mm9 genome assemblies, respectively. Up to 4 mismatches were allowed and bowtie (v0.12.8) was specified as the aligner.
Methylation was called using default parameters of BS-Seeker2.
Genome_build: sacCer3
Supplementary_files_format_and_content: wig
|
| |
|
| Submission date |
Mar 15, 2015 |
| Last update date |
May 15, 2019 |
| Contact name |
matteo pellegrini |
| E-mail(s) |
matteop@mcdb.ucla.edu
|
| Phone |
310 825-0012
|
| Organization name |
UCLA
|
| Street address |
610 charles young drive east
|
| City |
los angeles |
| State/province |
CA |
| ZIP/Postal code |
90095 |
| Country |
USA |
| |
|
| Platform ID |
GPL13821 |
| Series (2) |
| GSE66905 |
In vivo targeting of de novo DNA methylation by histone modifications in yeast and mouse (Bisulfite-Seq) |
| GSE66911 |
In vivo targeting of de novo DNA methylation by histone modifications in yeast and mouse |
|
| Relations |
| BioSample |
SAMN03417812 |
| SRA |
SRX957105 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM1634057_3b_exp.wig.gz |
15.8 Mb |
(ftp)(http) |
WIG |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
|
|
|
|
 |
