 |
 |
GEO help: Mouse over screen elements for information. |
|
| Status |
Public on Apr 02, 2015 |
| Title |
INPUT_1 |
| Sample type |
SRA |
| |
|
| Source name |
yeast cell culture
|
| Organism |
Saccharomyces cerevisiae |
| Characteristics |
growth phase: stationary growth phase
strain - genotype: wild-type/W303
chip antibody: N/A
salt concentration used for the extraction (mm): 140
|
| 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 |
Chromatin immunoprecipitation experiments were conducted according to Kitada et al. (Kitada et al., 2012), with minor modifications. Briefly, 50 OD of yeast cells are crosslinked using 1% formaldehyde for 15 minutes at room temperature and quenched with glycine 125 mM for 5 minutes at room temperature. After two washes with ice-cold PBS, the cells are resuspended in yeast lysis buffer (with 140 mM NaCl for DNMT3b and RNApolII or 500 mM NaCl for histone post-translational modifications) and the same volume of acid-washed glass beads. We disrupted the cells by vortexing for 5 minutes in a Disruptor Genie at 4°C and incubating in iced-water for 2 minutes. We repeated the cycle an additional five times. We collected the lysate by centrifugation after creating a hole on the bottom of the tube with a 25-G needle. We transferred a fraction of the lysate into a microTube (AFA filter – Covaris) and proceeded with the sonication using the Covaris S2 system according to the following parameters: 14 cycles of 30 sec ON, 30 sec OFF; Duty Cycle=5%; Intensity=5%; Cycles/Burst=200. The sonicated lysate is clarified via centrifugation and 50 μl of the supernatant are incubated over night at 4°C with a specific antibody (Supplementary File 6, Panel D). 10 μl of the clarified lysate are used as input control. The next day, immunoprecipitations are incubated 2 hours at 4°C with Protein A Dynabeads. Each wash is performed twice in the following order: low-salt buffer (50mM HEPES pH 7.5, SDS 0.1%, 1% Triton X-100, 0.1% Deoxycholate, 1mM EDTA, 140mM NaCl), high salt buffer (50mM HEPES pH 7.5, SDS 0.1%, 1% Triton X-100, 0.1% Deoxycholate, 1mM EDTA, 500 mM NaCl), LiCl buffer (10 mM Tris-HCl pH 8, 250 mM LiCl, 5mM EDTA, 1% Triton-X, 0.5% NP-40), TE buffer (100 mM Tris-HCl pH 8, 10 mM EDTA). Elution is performed at 65°C with TE/SDS buffer (100 mM Tris-HCl pH 8, 10 mM EDTA, 1% SDS). Tubes containing the eluted immunoprecipitations and input controls (additioned of TE/SDS buffer) are incubated overnight at 65°C to reverse the cross-links. RNase treatment is performed at 37°C for 1 hour, followed by a proteinase K treatment for 1 hour at 60°C. Each reaction is then purified using 1.8 volumes of AMPure XP beads according to manufacturer’s instructions.
Libraries were prepared with Ovation Ultralow DR kit (Nugen Technologies) starting from 1 ng of purified DNA according to the protocol.
|
| |
|
| Library strategy |
ChIP-Seq |
| Library source |
genomic |
| Library selection |
ChIP |
| Instrument model |
Illumina HiSeq 2000 |
| |
|
| Data processing |
Obtained reads were first mapped against the yeast (sacCer3) using bowtie v0.12.8 (Langmead et al., 2009)
Aligned reads were processed according to Ferrari et al. (Ferrari et al., 2012)
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) |
| GSE66909 |
In vivo targeting of de novo DNA methylation by histone modifications in yeast and mouse (ChIP-Seq yeast) |
| GSE66911 |
In vivo targeting of de novo DNA methylation by histone modifications in yeast and mouse |
|
| Relations |
| BioSample |
SAMN03417844 |
| SRA |
SRX957134 |
| Supplementary data files not provided |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data not applicable for this record |
|
|
|
|
 |
