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| Status |
Public on May 16, 2023 |
| Title |
Calibrated Scc1 ChIP-seq for isw1Δ INPUT |
| Sample type |
SRA |
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| Source name |
W303
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| Organisms |
Schizosaccharomyces pombe; Saccharomyces cerevisiae |
| Characteristics |
cell line: W303
antibody: input
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| Growth protocol |
~3 x 108 of S.cerevisiae cells expressing Scc1-9Pk were mixed with ~1 x 108 S.pombe cells expressing Rad21-6HA, fixed with 1% formaldehyde for 15 min at RT and then rocked for 5 min with 150 mM glycine. Next, cells were collected by centrifugation, washed once with TBS buffer and resuspended in 600 μl of FA-lysis buffer (50 mM HEPES-KOH pH 7.5, 140 mM NaCl, 1 mM EDTA, 0.1% sodium deoxycholate, 1% Triton X, protease inhibitors [Sigma-Aldrich, P8215], 1 mM PMSF). Cells were lysed by 4 rounds of bead-beating at 6500 rpm for 30 s. Next, whole cell extracts were transferred to new tubes and sonicated to yield an average DNA size of 500 bp followed by 30 min centrifugation at 15 000 rpm. Cleared lysates were moved to new tubes and 2% of the lysates were kept as an input sample. For the IP reaction, 7 μl of anti-Pk antibodies (Bio-Rad, MCA1360G) and 3 μl of anti-HA (Roche,11583816001) were added to the lysates and incubated overnight at 4°C on a rotator wheel. Next day, DNA-protein complexes were captured with Protein G Dynabeads (Thermo Fisher, 10003D) and sequentially washed with FA-lysis buffer, FA-500 buffer (50 mM HEPES–KOH pH 7.5, 500 mM NaCl, 1% Triton X-100, 0.1% sodium deoxycholate, 1 mM EDTA), LiCl wash buffer (10 mM Tris–HCl pH 8.0, 250 mM LiCl, 0.5% NP-40, 0.5% sodium deoxycholate, 1 mM EDTA) and TE (10 mM Tris–HCl pH 7.5, 1 mM EDTA) on a rotator for 5 min each time.
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
Beads and input samples were resuspended in elution buffer (50 mM Tris–HCl pH 7.5, 1% SDS, 10 mM EDTA) and incubated at 65°C for 2 h on a thermal shaker. Next, beads were removed and the IP eluates and input samples were incubated at 65°C overnight. Next day, all the samples were treated with RNAse A for 1 h at 37°C followed by 2h incubation at 65°C in the presence of Proteinase K. Finally, DNA was recovered using a Monarch PCR & DNA Cleanup Kit (New England BioLabs, T1030L).
DNA libraries were constructed using NGS DNA Library Prep Set (Novogene, PT004).
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| Library strategy |
ChIP-Seq |
| Library source |
genomic |
| Library selection |
ChIP |
| Instrument model |
Illumina NovaSeq 6000 |
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| Data processing |
The adapter sequences and low-quality bases were trimmed off using Skewer.
Filtered reads were mapped against the target genome (S. cerevisiae S288C assemblySacCer3) and against the spike-in genome (S. pombe assembly ASM294v2) using BWA.
Unmapped reads, rDNA regions, mitochondrial DNA and duplicates were filtered out using Samtools.
Peaks were called using MACS2.
In order to visualize mapped reads, bigWig files were created using deepTools.
The ChIPs were normalized against the input with Counts Per Million (CPM) method.
Samtools was used to count reads mapped to SacCer3 and ASM294v2 only, and these values were used to calculate the Occupancy Ratio value.
Plots were created around the midpoint of CENs.
Assembly: SacCer3, ASM294v2
Supplementary files format and content: bigWig
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| Submission date |
May 11, 2023 |
| Last update date |
May 17, 2023 |
| Contact name |
Ireneusz Bernard Litwin |
| E-mail(s) |
[email protected]
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| Phone |
+48663491371
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| Organization name |
University of Wroclaw
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| Department |
Academic Excellence Incubator - Research Centre for DNA Repair and Replication
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| Lab |
Karol Kramarz's lab
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| Street address |
Kanonia 6/8
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| City |
Wrocław |
| ZIP/Postal code |
50-328 |
| Country |
Poland |
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| Platform ID |
GPL29170 |
| Series (1) |
| GSE232315 |
Isw1 contributes to cohesin association with centromeres and pericentromeric regions |
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| Relations |
| BioSample |
SAMN35047803 |
| SRA |
SRX20296468 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM7326248_isw1_Input.bw |
9.5 Mb |
(ftp)(http) |
BW |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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