|
| Status |
Public on Dec 18, 2015 |
| Title |
no OE 35 min replicate 2 IP |
| Sample type |
SRA |
| |
|
| Source name |
S. cerevisiae
|
| Organism |
Saccharomyces cerevisiae |
| Characteristics |
strain: no OE ( WT pGAL)
drug: No Drug
time point: S-phase pulse 20-35 min
barcode (5'): BC1
chip antibody: BrdU antibody (Invitrogen, catalog#033900)
|
| Treatment protocol |
Cells were blocked in G1 by resuspension (at O.D. ~0.5) in fresh YEP+2% raffinose +7.5 nM α-factor at 25°C for 3 hrs. For induction, cells were resuspended in YEP+2% galactose +7.5 nM α-factor at 25°C for 2 hrs. Cultures were released into S-phase by resuspension (at O.D. ~1.0) in fresh YEP+2% galactose +200 µg/mL Pronase E (Sigma-Aldrich, P5147) and sonicated to disperse cells. Early S-phase analysis of replication was performed by including 0.2M HU (Sigma-Aldrich, H8627) and BrdU at 400 µg/mL (Sigma-Aldrich, B5002) for 60 min at 25°C. For the time course experiment, BrdU was used at 800 µg/mL.
|
| Growth protocol |
For late G1 block, induction, and release, cells (pre-selected on SD-URA for plasmid as appropriate) were inoculated into YEP+2% raffinose at 25°C and grown to mid-log phase
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
Genomic DNA was isolated with standard phenol chloroform extraction
Genomic DNA isolation and shearing by sonication has been described previously (Knott et al., 2012) 1 µg total genomic DNA (sheared to ~300 bp average) was end-repaired and ligated with a barcoded, Illumina-compatible adapter using NEBNext Ultra End Repair/dA-Tailing Module (E7442) and NEBNext Ultra Ligation Module (E7445) (Dunham and Friesen, 2013). AMPure beads (Beckman Coulter Cat#A63880) were used to isolate the genomic DNA and its concentration was determined by spectroscopy (Nanodrop). Equal amounts of multiple such barcoded DNA samples were pooled; 20 ng was set aside as “Input” and 1 µg of this pool was subject to BrdU-IP as described previously (Knott et al., 2012). The IP and Input DNA samples were PCR-amplified separately with indexed Illumina-compatible primers. Amplified DNA was isolated using AMPure beads and validated and quantified on a Bioanalyzer, and pooled.
Quantitative BrdU-IP Seq (QBU)
|
| |
|
| Library strategy |
OTHER |
| Library source |
genomic |
| Library selection |
other |
| Instrument model |
Illumina NextSeq 500 |
| |
|
| Description |
BrdU labeled genomic DNA immunoprecipitated from galactose induced culture released into early S-phase at the indicated timepoint
QBU_Time_Course.txt
|
| Data processing |
QBU Samples:
fastq-multx by ea-utils was used to split barcodes
Barcode split PE files were aligned to S. cerevisiae reference genome R64-1-1 using Bowtie2
Aligned files were sorted and binned into 50 bp non-overlapping bins using Samtools/Bedtools
For QBU samples, IP bins were divided by their corresponding Input (Total) Sample Bins to yield normalized data
RNA-Seq Samples:
Samples were aligned to S. cerevisiae reference genome R64-1-1 with Tophat2 using a known transcript file (.gtf)
Samples were next subjected to the Cufflinks transcript assembly and differential experession pipeline including Cuffdiff to call differentially expressed transcripts (FDR <= 0.01)
Genome_build: R64-1-1
Supplementary_files_format_and_content: .bed file
|
| |
|
| Submission date |
Jul 17, 2015 |
| Last update date |
May 15, 2019 |
| Contact name |
Jared M Peace |
| Organization name |
University of Southern California
|
| Department |
Molecular and Computational Biology
|
| Lab |
Oscar Aparicio
|
| Street address |
1050 Childs Way RRI 203
|
| City |
Los Angeles |
| State/province |
CA - California |
| ZIP/Postal code |
90089 |
| Country |
USA |
| |
|
| Platform ID |
GPL19756 |
| Series (2) |
| GSE71050 |
Quantitative BrdU immunoprecipitation method demonstrates that Fkh1 and Fkh2 are rate-limiting activators of replication origins that reprogram replication timing in G1 phase (BrdU) |
| GSE71052 |
Quantitative BrdU immunoprecipitation method demonstrates that Fkh1 and Fkh2 are rate-limiting activators of replication origins that reprogram replication timing in G1 phase |
|
| Relations |
| BioSample |
SAMN03877154 |
| SRA |
SRX1100209 |
