 |
 |
GEO help: Mouse over screen elements for information. |
|
| Status |
Public on Aug 28, 2018 |
| Title |
y1431: dpb4∆ H3K4me3 ChIP-ssSeq |
| Sample type |
SRA |
| |
|
| Source name |
whole cell
|
| Organism |
Saccharomyces cerevisiae |
| Characteristics |
media: YPD+HU
cell cycle stage: S
chip antibody: Anti-H3K4me3 Antibody (ab8580)
|
| Growth protocol |
To synchronize cells at G1, alpha factor was added at cell density OD600=0.4-0.5 to achieve a final concentration of five µg/mL for yeast strains. After three hours arrest at 25°C, the cells were released to fresh YPD medium with or without 200 mM HU. For the histone H3 Recombination-induced Tag Exchange (RITE) experiments, yeast cells were grown overnight in YPD in the presence of hygromycin B (200 μg/mL, Invitrogen). The cells were then diluted 1:10 into two fresh YPD flask (one with, the other without hygromycin B) and incubated for 32 hours (G0 state). The cells were centrifuged by 3000 rpm for 10 minutes. The cells cultured with hygromycin B were transferred to another flask containing depleted medium without hygromycin B. Recombination was induced by the addition of 1 μM β-estradiol (E-8875, Sigma-Aldrich) and the cells were cultured for additional 16 hours. Subsequently, these cells were diluted 1:25 in fresh YPD media containing 200 mM HU and 400ul/ml BrdU. After incubation at 25 C for 90 minutes, samples were collected for ChIP and eSPAN.
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
All ChIP and eSPAN assays were performed as described previously except that Mnase digestion was used for chromatin fragmentation in order to map nucleosomes. Briefly, the samples were incubated with 1% fresh-made formaldehyde at 25 °C for 20 min, and then quenched with 0.125M glycine for five min. The cells wer harvested and lysed with glass beads in ChIP lysis buffer (50 mM HEPES, pH 8.0, 150 mM NaCl, 2 mM EDTA, 1% Triton X-100, and 0.1% sodium deoxycholate). The resulting cell and chromatin pellet was washed with NP buffer (1.6 M sorbitol, 2 mM CaCl2, 5mM MgCl, 50 mM NaCl, 14 mM β-mercaptoethanol, 10 mM Tris-HCl (pH 7.4), 0.075% NP-40, 5 mM spermidine). Proper amounts of Mnase was added to ensure that majority of chromatin are mono- and di-nucleosomes with incubation at 37 °C for 20 min . The reacation was terminated by adding 5ul 0.5M EDTA. Further, ¼ volume 5X ChIP lysis buffer was added into dilute thedigested chromatin. The lysates was incubated in ice for 30 minutes. After clarification of lysates by centrifugation, soluble chromatin (mainly mononucleosomes) was immunoprecipitated with a specific antibody including anti-H3K4me3 antibody (ab8580), anti-H3K56ac antibody (6), anti-HA antibody (12CA5) or anti-T7 (A190-117A BETHYL)). ChIP DNA was recovered using Chelex-100. The ChIP or total DNA was denatured at 100°C for five min and was immediately transferred to ice bath for five minutes. The DNA was diluted at least ten times with BrdU IP solution (PBS, 0.0625% Triton X-100(v/v), 6.7 g/mL Escherichia. coli tRNA, 0.67 ul/mL BrdU antibody (BD Bioscience)). After the samples were incubated at 4°C for two hour, 20 uL protein G beads (GE Healthcare) were added. After incubation at 4°C for another hour, the protein G beads was washed extensively. DNA were eluted using 100 uL 1XTE buffer plus 1% SDS and incubation at 65 °C for 15 min. Eluted DNA was purified using a Qiagen Mini-elute kit.
We used the ssDNA library preparation kit to prepare library (Cat no. 10096 Swift Bioscience). The ssDNA libraries were sequenced using the paired-end method by different sequencing platform over the years including Illumina NextSeq 500 or HiSeq 2000 machine at Mayo Clinic or Columbia University.
|
| |
|
| Library strategy |
ChIP-Seq |
| Library source |
genomic |
| Library selection |
ChIP |
| Instrument model |
Illumina NextSeq 500 |
| |
|
| Data processing |
Sequence reads were mapped back to the yeast reference genome (sacCer3) using Bowtie2 software. Only consistent pair-end reads were chosen for further analysis.
The sequence reads mapped to the Watson and Crick strands of the reference genome were separated by in-house Perl programs and then genome-wide reads coverage was calculated by BEDTools and in-house Perl programs.
To obtain the nucleosome occupancy, DNA fragments from 120 to 170 bp in size determined by the paired-reads were firstly selected for calculation. The length of DNA fragments were then adjusted to half of the nucleosome size (74bp) to increase the signal-to-noise ratio. Finally, the count of adjusted DNA fragments covering each base pair in the genome were used to calculate the nucleosome occupancy.
To identify BrdU-IP-ssSeq peaks, the sequence reads of both Watson and Crick strands were merged and used to call peaks using SICER software with false discovery rate (FDR) cut-off 0.01.
Genome_build: sacCer3
Supplementary_files_format_and_content: bigwig files were generated for Watson and Crick strand, scores represent the normalization reads density.
|
| |
|
| Submission date |
Mar 30, 2018 |
| Last update date |
Aug 28, 2018 |
| Contact name |
zhiguo zhang |
| E-mail(s) |
[email protected]
|
| Phone |
212-851-4936
|
| Organization name |
Columbia University
|
| Department |
Pediatric and Genetics and Development
|
| Lab |
Irving Cancer Research Center
|
| Street address |
1130 St. Nicholas Avenue
|
| City |
New York |
| State/province |
NY |
| ZIP/Postal code |
10032 |
| Country |
USA |
| |
|
| Platform ID |
GPL19756 |
| Series (1) |
| GSE112522 |
Asymmetric partition of parental histone (H3-H4)2 tetramers onto replicating DNA strands |
|
| Relations |
| BioSample |
SAMN08821984 |
| SRA |
SRX3865874 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM3072035_y1431_minus.bw |
17.8 Mb |
(ftp)(http) |
BW |
| GSM3072035_y1431_plus.bw |
17.8 Mb |
(ftp)(http) |
BW |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
|
|
|
|
 |
