 |
 |
GEO help: Mouse over screen elements for information. |
|
| Status |
Public on Jul 16, 2021 |
| Title |
mrc1D_rad9_IAA brdu rep2 |
| Sample type |
SRA |
| |
|
| Source name |
mrc1D_rad9_IAA brdu
|
| Organism |
Saccharomyces cerevisiae |
| Characteristics |
experiment: BrdU
condition: with IAA
genotype: mrc1D rad9
chip antibody: BrdU antibody (BD Bioscience)
|
| Treatment protocol |
ChIP-ssSeq and BrdU-IP-ssSeq experiments were performed as described previously (Gan et al., 2017)
|
| Growth protocol |
Yeast cells grown to OD600=0.4-0.5 in yeast extract peptone + 2% dextrose (YPD) were synchronized with a-factor (5 mg/ml, synthesized by EZBiolab) for 1.5 hr at 25°C, adding additional 5 mg/ml a-factor after 1.5 hr. G1-arrested cells were released into YPD medium at 30°C containing 400 mg/ml bromodeoxyuridine (BrdU, Sigma B5002) and 0.2M hydroxyurea (HU, Chem-Impex 24533). 45 minutes after release into S phase at 30°C, cell fixation was performed with 1% paraformaldehyde for 20 minutes at room temperature followed by the quenching with 0.125M glycine for 5 minutes at room temperature.
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
Input DNA obtained from the Chelex-100 extraction was used for BrdU immunoprecipitation. Briefly, DNA was denatured at 95°C for 5 minutes followed by incubation in an ice-water bath for 5 minutes. Samples were then diluted 10 fold with BrdU IP buffer solution (1XPBS, 0.0625% Triton X-100(v/v), 6.7 mg/mL Escherichia coli tRNA, 0.40mL/mL BrdU antibody (BD Bioscience)). After a 2 hr incubation at 4°C, 20 mL of protein G Sepharose beads (GE Healthcare) were added followed by an additional 1 hr incubation at 4°C. Protein G beads were extensively washed and DNA was eluted with 100 mL Tris-EDTA buffer containing 1% SDS at 65°C for 15 minutes.
The eluted DNA was purified with Minelute PCR kit (QIAGEN) and sequenced after ssDNA library preparation using the Accel-NGS 1S Plus DNA library kit (Swift BioSciences). Samples were pooled and sequenced using paired-end sequencing on Illumina platforms at Columbia University Genome Center.
|
| |
|
| Library strategy |
OTHER |
| Library source |
genomic |
| Library selection |
other |
| Instrument model |
Illumina HiSeq 2000 |
| |
|
| Data processing |
Library strategy: BrdU-Seq
Reads were mapped using bowtie2.
Read coverage in each bin was calculated using Deeptools. Histone bias was computed from trimmed, mapped, unique reads in each bin as Bias=(W-C)/(W+C), where W and C are the number of reads mapped on Crick and Watson strand in each bin, respectively.
Genome_build: For yeast, reads were mapped to saccer3.
Supplementary_files_format_and_content: bw files, the score represents reads coverage at each genomic position. Read coverage on plus strand, minus strand and total were calculated seperately.
|
| |
|
| Submission date |
Apr 14, 2021 |
| Last update date |
Jul 16, 2021 |
| Contact name |
Xu Hua |
| E-mail(s) |
[email protected]
|
| Organization name |
Columbia University
|
| Department |
Institute for Cancer Genetics
|
| Lab |
Zhiguo Zhang
|
| Street address |
1130 St Nicholas Ave
|
| City |
New York |
| State/province |
New York |
| ZIP/Postal code |
10032 |
| Country |
USA |
| |
|
| Platform ID |
GPL13821 |
| Series (1) |
| GSE172093 |
A mechanism of coupling leading and lagging strand DNA synthesis under replication stress in budding yeast |
|
| Relations |
| BioSample |
SAMN18742446 |
| SRA |
SRX10602400 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM5241692_rad53_14_minus.bw |
3.6 Mb |
(ftp)(http) |
BW |
| GSM5241692_rad53_14_plus.bw |
3.6 Mb |
(ftp)(http) |
BW |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
|
|
|
|
 |
