|
| Status |
Public on Aug 25, 2016 |
| Title |
Nascent DNA S-phase time course 2 |
| Sample type |
SRA |
| |
|
| Source name |
Nascent DNA S-phase time course
|
| Organism |
Saccharomyces cerevisiae |
| Characteristics |
strain: wt yeast derived from W303 with the h-ENT1 nucleotide transporter and HPV-Thymidine kinase
cell cycle: S-phase
edu incubation: from beginning of release from G1 arrest
thymidine incubation (min): NA
time point: 32min after release, nascent S-phase chromatin
|
| Treatment protocol |
Cells were fixed in 1% formaldehyde.
|
| Growth protocol |
For the synchronization experiment cells were grown over night at 30°C in Synthetic Complete- URA + Dextrose media to OD 0.3. After 3.75hrs of incubation at 30°C with alpha factor (0.25μg/ml), cells were pelleted and transferred into preheated and premixed SCD-URA+ 10μM EdU(Carbosynth), with freshly added 20μg/ml pronase (Sigma). 200ml aliquots were taken at 25, 32, 40, 47 and 55 min after release. Time points were fixed with 1% formaldehyde.
In the EdU-Thymidine pulse chase experiments, cells were grown in SCD-URA over night at 30°C to an OD of 1.0. The next day the culture was diluted to OD=0.25 and cells were allowed to double once. Cell pellets were then transferred to preheated and premixed SCD-URA+ 10μM EdU media. Thymidine (to a final concentration of 5mM) was added after 5,10 or 20min incubation with EdU at 30°C and incubated for another 5 or 10 min. Cells were then pelleted and transferred into fresh media with 5mM Thymidine (and 15μg/ml nocodazole 5(Sigma) or 3μg/ml thiolutin (Abcam) when indicated) and 200ml aliquots were taken at indicated time points (after the Thymidine incubation) and fixed as above.
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
Cells were washed in water, cell walls were broken with bead beating and spheroplasts were treated with micrococcal nuclease. EdU from isolated nascent DNA fragments was biotinylated using Click chemistry and nascent DNA was purified with streptavidin coated beads. An input DNA aliquot was set aside prior to biotinylation.
Nascent and input DNA samples, were treated with calf intestinal phosphatase (NEB), blunt-ended, A-tailed, and ligated (Epicentre) to Illumina genomic PE adaptors with custom 5bp inline barcodes (first 5bp in raw data read), and PCR amplified and size selected by gel purification. For nascent libraries, the nascent strand was separated from its complement with primer extension in the presence of dUTP and the dUTP strand was then digested with the USER enzyme cocktail prior to PCR amplification. All the steps for nascent libraries were done on streptavidin coated magnetic beads.
|
| |
|
| Library strategy |
MNase-Seq |
| Library source |
genomic |
| Library selection |
MNase |
| Instrument model |
Illumina Genome Analyzer |
| |
|
| Description |
MNase digest time course followed by nascent DNA purification by EdU-biotin pulldown from synchronized cells in S-phase.
Strand-specific MNase-seq.
Processed data file: Sync_EdU_input_tss_aligned.txt
|
| Data processing |
Illumina OLB-1.8.0/CASAVA_v1.7.0 were used for base calling.
Time points were separated by barcode and the 5bp barcode was removed from the read sequence.
Reads were mapped to the yeast S288C genome using BLAT.
Paired end read count distribution in 1bp windows was determined separately for Watson (marked as F in the processed data files) and Crick (marked as R in the processed data files) strands.
The average read count value was calculated and the data were normalized to one.
Genome_build: S288C (S. cerevisiae)
Supplementary_files_format_and_content: Tab-delimited text files of nucleosome occupancy for every gene at indicated time points (lines), showing every 20th bp from -500bp to the end of the gene coding sequence surrounding the transcription start site (marked as tss). Occupancy data start after the GWEIGHT header.
|
| |
|
| Submission date |
Oct 16, 2015 |
| Last update date |
May 15, 2019 |
| Contact name |
Marta Radman-Livaja |
| E-mail(s) |
mrl5374@gmail.com
|
| Phone |
+33434359667
|
| Organization name |
CNRS
|
| Department |
IGMM
|
| Street address |
1919 route de Mende
|
| City |
Montpellier |
| ZIP/Postal code |
34293 |
| Country |
France |
| |
|
| Platform ID |
GPL9134 |
| Series (1) |
| GSE74090 |
Dynamics of chromatin maturation after genome replication |
|
| Relations |
| BioSample |
SAMN04191409 |
| SRA |
SRX1341458 |
