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| Status |
Public on May 23, 2024 |
| Title |
ChIP_YPD_repeat 2 |
| Sample type |
SRA |
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| Source name |
W303ScSef1TAPNB1-1
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| Organism |
Saccharomyces cerevisiae |
| Characteristics |
strain: W303ScSef1TAPNB1-1
genotype: MATa his3-11,15 trp1-1 ura3-1 ade2-1 leu2-3,112 can1-100 SEF1-TAP::FRT
condition: YPD, 30°C
time: 4.5 h
growth phase: log-phase, OD = ~1.0
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| Treatment protocol |
After 4.5 h of incubation at 30°C, cultures of log phase cells (1 OD600/ml in YPD and 0.4 OD600/ml in YPGly) were fixed with 1% formaldehyde at 25°C for 15 min with shaking at 180 rpm and then quenched with 125 mM glycine at 25°C for 10 min with shaking at 180 rpm. All the following steps were performed in an ice-cold or 4°C environment. Cells were harvested, washed twice with TBS (20 mM Tris-Cl, pH 7.5; 150 mM NaCl), and stored at −80°C until use.
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| Growth protocol |
Cells of W303ScSef1TAPNB1-1 (SEF1-TAP) were grown in the YPD medium at 30°C overnight, subsequently diluted into each indicated medium in a cell density of 0.2 OD600/ml, and then incubated at 30°C for 4.5 h.
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
For breaking cells, 1500 and 500 OD600 of cells from YPD and YPGly, respectively, were thawed and resuspended in FA buffer (50 mM HEPES, pH 7.5; 140 mM NaCl; 0.1% SDS; 1 mM EDTA; 1% Triton X-100; 0.1% Deoxycholate, Na salt) with 1/100 PIC (Protease inhibitor cocktail Set IV, in DMSO; 539136, Merck) and 1 mM phenylmethanesulfonyl fluoride (PMSF, P7626, Sigma) at a concenration of 500 OD600/4ml. Every 4-ml cell suspension was divided into four 2-ml breaking tubes (Biospec, 10832, Microtube 2 ml with cap) containing 1 ml volume of glass beads (11079105, BioSpec Products). The lysis was performed by three cycles of 5-min beating followed by 1-min chilling on ice (Biospec Mini-BeadBeater-16). Cell lysates and glass beads were separated by punching a hole on the bottom of the tube with a red-hot 18G needle and slow centrifugation (500 g, 3 min, 4°C; Eppendorf 5810R centrifuge, A-4-62 rotor). The collected lysates were combined into a 15-ml centrifuge tube and washed with two cycles of 5 ml FA buffer followed by centrifugation (12K rpm, 5 min, 4C; Eppendorf 5810R centrifuge, F-34-6-38 rotor). The washed lysates were completely resuspended in 2 ml FA buffer with 1/100 PIC and 1 mM PMSF in a new 15-ml centrifuge tube (CFT011150, BIOFIL). Chromatin was sheared by sonication in a Bioruptor water bath sonicator (Diagenode) with 15 ml tube-chip unit (High intensity; 30 sec on, 30 sec off, 15 min/cycle; total 3 cycles) at 4°C. The sheared lysate was centrifuged at 12000 g for 10 min at 4°C (Eppendorf 5810R centrifuge, F-34-6-38 rotor). About 25 ml of supernatant corresponding to 5 OD600 of cells was collected, mixed with 200 ml TES buffer (50 mM Tris-Cl, pH 8; 10 mM EDTA, pH 8; 1% SDS) plus 175 ml TE buffer (10 mM Tris-Cl; 1 mM EDTA, pH 8), and stored at -20°C as the input-DNA. Immunoprecipitation was performed by incubating all the rest sheared chromatins with 300 ml Dynabeads® Pan Mouse IgG (11041, human anti-mouse IgG, Invitrogen) in 6 ml of FA buffer with 1/100 PIC and 1 mM PMSF. The binding was performed in a 15-ml tube with mixing using an end-over-end rotator (Fntelli-Mixer, RM-2L, F1 mode, 30 rpm) at 4°C for 16 h. The magnetic beads were anchored with DynaMagTM-2 Magnet and the bound complexes were washed 4 times with 1 ml of each wash buffer for 5 min [FA buffer × 1, high-salt FA buffer (50 mM HEPES, pH 7.5; 500 mM NaCl; 0.1% SDS; 1 mM EDTA; 1% Triton X-100; 0.1% Deoxycholate, Na salt) × 1, DOC buffer (10 mM Tris·Cl; 1 mM EDTA, pH 8; 25 0mM LiCl; 0.5% IGEPAL® CA-630; 0.5% Deoxycholate, Na salt) × 1, and TE buffer × 1] with mixing (Fntelli-Mixer, RM-2L, F1 mode, 30 rpm) at room temperature. The bound complexes were eluted twice by heating at 65°C in 200 ml TES buffer for 20 min and in 200 ml TE buffer for 10 min. Two eluates were pooled as the ChIP-DNA. To remove RNA, both the input-DNA and ChIP-DNA (~400 ml each) were treated with 5 ml of 10 mg/ml RNase A (R5503, Sigma; 100 mM Tris-Cl, pH 7.4) at 37°C for 30 min. For de-crosslinking, both the RNase-treated input-DNA and ChIP-DNA were treated with 40 ml of 20 mg/ml Proteinase K (1.24568.0500, Merck; in 50 mM Tris-Cl, pH 8) at 42°C for 1 h and then 65°C for 16 h. De-crosslinked DNA was purified using the QIAquick DNA Purification Kit (28106, Qiagen) according to the manufacturer's instruction, with a modification of a two-cycle wash step using the PE buffer. Final ChIP-DNA and input-DNA were eluted with 50 ml EB buffer. DNA concentrations were measured by using the Qubit™ dsDNA HS Assay Kit (Q32854, Invitrogen by Thermo Fisher Scientific).
ChIP-seq analysis was performed from three biological replicates of YPD- and YPGly-grown cells. The average fragment length of sonicated fragments was 150–1000 bp. For each condition, libraries were prepared from >3 ng of ChIP-DNA and input-DNA using the KAPA LTP Library Preparation Kit for Illumina® platforms (KK8232, KAPA Biosystems by Roche) according to the manufacturer’s instruction. Notably, after adapter ligation, double-sided size selection between 250–450bp for adapter-ligated fragments was performed using the KAPA Pure Beads (KK8000/07983271001, KAPA Biosystems by Roche). Size-selected fragments were then PCR-amplified for 12 cycles. The size of each library was assessed using a Bioanalyzer 2100 instrument (Agilent Technologies) with the High Sensitivity DNA Kit (Agilent Technologies). The concentration of each library was quantified by using the Qubit™ dsDNA HS Assay Kit (Q32854, Invitrogen by Thermo Fisher Scientific) and qPCR. Single-read sequencing (75 bp) or paired-end sequencing (75 bp × 2) of the libraries was performed using the NextSeq 500/550 high output reagent kit V2_75 cycle (FC-404-2005, Illumina) on an Illumina NextSeq500 sequencer.
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| Library strategy |
ChIP-Seq |
| Library source |
genomic |
| Library selection |
ChIP |
| Instrument model |
Illumina NextSeq 500 |
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| Description |
paired with ScD2 Input for peak calling
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| Data processing |
All sequencing protocols were carried out as per the manufacter's instructions using the Illumina NextSeq500 and NextSeq software: base calling was performed using NextSeq Control Software NCS 2.2.0 and Real-time Analysis software RTA 2.4.11 to generate BCL files. The Illumina bcl2fastq2 Conversion Software v2.17 was used to demultiplex sequencing data and convert BCLfiles into FASTQ files.
The quality control and adaptor trimming of sequencing reads were processed by FastQC version 0.11.8 (https://www.bioinformatics.babraham.ac.uk/projects/fastqc/) and Trimmomatic version 0.36 with the parameters “2:30:10 LEADING:3 TRAILING:3 SLIDINGWINDOW:4:15 HEADCROP:3 CROP:68”.
Trimmed reads were mapped to the S. cerevisiae S288C genome using Bowtie2 version 2.3.3.
Bowtie2 mapped BAM files were detected for the ChIP peaks using SPP version 1.13 with a false discovery rate (FDR) of 0.05.
The Sef1 binding peaks were compared and merged among three or four biological replicates by Diffbind version 2.10.0. Only peaks detected in all three biological repeats were used for the downstream analysis.
The text files, wig files, and narrowPeak files were generated from SPP. The bigwig files are generated by deepTools.
Assembly: Saccharomyces cerevisiae S28C genome (R64-2-1)
Supplementary files format and content: Text files with binding positions, wig files, narrowPeak, bigwig files
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| Submission date |
Mar 25, 2024 |
| Last update date |
May 23, 2024 |
| Contact name |
Po-Chen Hsu |
| E-mail(s) |
godshi2006@gmail.com
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| Organization name |
Academia Sinica
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| Department |
Institute of Molecular Biology
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| Lab |
N411 JYL lab
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| Street address |
128 Academia Road, Section 2, Nankang
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| City |
Taipei |
| ZIP/Postal code |
11529 |
| Country |
Taiwan |
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| Platform ID |
GPL19756 |
| Series (1) |
| GSE262389 |
The protein moonlighting dominates the phenotypic divergence of the Sef1 transcriptional regulatory networks in yeasts |
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| Relations |
| BioSample |
SAMN40612043 |
| SRA |
SRX24057039 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM8164309_ScD2_ChIP_R1.bigWig |
11.8 Mb |
(ftp)(http) |
BIGWIG |
SRA Run Selector |
| Raw data are available in SRA |
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