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| Status |
Public on Jan 06, 2022 |
| Title |
s1_H3_WT_0 |
| Sample type |
SRA |
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| Source name |
s1_H3_WT_0
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| Organism |
Saccharomyces cerevisiae |
| Characteristics |
genotype: WT
protein: H3_input
condition: control
cell source: CEN.PK cells
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| Treatment protocol |
To perform glucose starvation, we quickly replaced the media by spinning down the cells, washing the pellets with pre-warmed S media, and then resuspending cells in the same volume of warmed S media. Samples were collected at indicated times. Glucose replenishment was carried out at a final concentration of 2% glucose. All centrifugations were performed at 3000 rpm, 2 min at room temperature.
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| Growth protocol |
Strains of interest were grown in SD medium overnight. Cells from overnight cultures were inoculated into fresh SD to 0.2 optical density (OD)/ml and grown at least two generations to log phase (OD600 ~1).
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
ChIP was performed as described previously (Cai et al., 2011). Briefly, 100 OD600 units of cells were fixed in 1% formaldehyde for 15 min and then quenched in 125 mM glycine for 10 min. Cell pellets were washed twice with a buffer containing 100 mM NaCl, 10 mM Tris-HCl pH 8.0, 1 mM PMSF, and 1 mM benzamide-HCl, and snap-frozen in liquid nitrogen. Frozen pellets were lysed in 0.45 mL ChIP lysis buffer (50 mM HEPES-KOH pH 7.5, 500 mM NaCl, 1 mM EDTA, 1% Triton X-100, 0.1% deoxycholate, 0.1% SDS,1 mM PMSF, 10 µM leupeptin, 5 µM pepstatin A, and Roche protease inhibitor cocktail) and lysed by bead-beating. Lysates were split to two tubes and sonicated for 16 cycles (30 sec on, 1 min off, high output) using the Bioruptor (Diagenode). The lysates were then cleared by two consecutive spins at 15,000 rpm for 10 min. For HA ChIP, lysate was incubated overnight with 10 µg antibody. For histone ChIP, lysate was diluted at 1:10 and incubated overnight with 2 µg antibody. 25 µl magnetic beads were added and incubated for an additional 1.5 h. Beads were then washed twice with ChIP lysis buffer, once with deoxycholate buffer (10 mM Tris-HCl, 0.25 M LiCl, 0.5% deoxycholate, 1 mM EDTA), and once with TE buffer (50 mM Tris-HCl pH 8.0, 10 mM EDTA). Elution was performed using TES buffer (50 mM Tris-HCl pH 8.0, 10 mM EDTA, and 1% SDS). Reverse crosslinking was performed at 65°C. An equal volume of TE containing 1.25 mg/mL proteinase K and RNase A was then added and incubated for 2 h at 37°C. DNA was then purified using the Qiagen PCR purification kit.
ChIP samples were prepared as described above in two biological replicates. We constructed sequencing libraries using KAPA Hyper Prep Kit from Kapa Biosystems. The quality and quantity of constructed libraries were determined using Agilent D1000 ScreenTape System and Invitrogen Qubit Fluorometer. The sequencing was performed by the UTSW Next Generation Sequencing Core Facility using the Illumina NextSeq 500 System.
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| Library strategy |
ChIP-Seq |
| Library source |
genomic |
| Library selection |
ChIP |
| Instrument model |
Illumina NextSeq 500 |
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| Data processing |
We use the BICF ChIP-seq analysis Workflow version 1.1.3 (Barnes et al., 2020). The workflow used trimgalore version 0.4.1 (Martin et al., 2011) on the raw reads to remove adapter sequences and to remove reads shorter than 35bp or with phred quality scores less than 20. Trimmed reads were then aligned to the yeast reference genome (S288C) using default parameters in BWA version 0.7.12 (Li and Durbin, 2009). The aligned reads were subsequently filtered for quality and uniquely mappable reads were retained for further analysis using Samtools version 1.3 (Li and Durbin, 2009) and Sambamba version 0.6.6 (Tarasov et al., 2015). Library complexity was measured using BEDTools version 2.26.0 (Quinlan and Hall, 2010) and meets ENCODE data quality standards (Landt et al., 2012). We determined ChIP enrichment compared to background using ‘plotFingerprint’ in deepTools version 2.5.0 (Ramirez et al., 2016). Relaxed peaks were called using MACS version 2.1.0 (Feng et al., 2012) with a p-value of 1x10-2 for each replicate, pooled replicates’ reads and pseudoreplicates. Peak calls from the pooled replicates that are either observed in all replicates, or in both pseudoreplicates were kept. Reads were counted at promoter regions using subread ‘featurecount’ (v1.4.6) (Liao et al., 2014) per gene ID against the same genome as above and used for subsequent analysis. Reads overlapping multiple genes were not discarded. Small-RNAs and genes with logcpm of < 10 were filtered out.
Genome_build: S288C
Supplementary_files_format_and_content: bigwig files
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| Submission date |
Jun 14, 2021 |
| Last update date |
Jan 06, 2022 |
| Contact name |
Wen-Chuan Hsieh |
| E-mail(s) |
[email protected]
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| Organization name |
UTSouthwestern Medical Center
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| Department |
Biochemistry
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| Lab |
Tu lab
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| Street address |
5323 Harry Hines Blvd
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| City |
Dallas |
| State/province |
Texas |
| ZIP/Postal code |
75390 |
| Country |
USA |
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| Platform ID |
GPL19756 |
| Series (2) |
| GSE178159 |
Glucose Starvation Induces a Switch in the Histone Acetylome for Activation of Gluconeogenic and Fat Metabolism Genes [ChIP-seq] |
| GSE178161 |
Glucose Starvation Induces a Switch in the Histone Acetylome for Activation of Gluconeogenic and Fat Metabolism Genes |
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| Relations |
| BioSample |
SAMN19696913 |
| SRA |
SRX11141065 |
| Supplementary data files not provided |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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