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| Status |
Public on Aug 23, 2012 |
| Title |
sba1_deletion_DNase-seq |
| Sample type |
SRA |
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| Source name |
BY4741_sba1Δ
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| Organism |
Saccharomyces cerevisiae |
| Characteristics |
strain: BY4741 (MATa)
genotype: sba1-/-
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| Growth protocol |
The strains used for the DNase-Seq experiments were the parent BY4741 (MATa) along with sba1Δ and gcn5Δ isolates (Open BioSystems). Yeast cells were grown in rich medium (YPD) supplemented with 2% glucose.
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
The DNase-Seq protocol was adapted from a previous method with minor modifications(NATURE METHODS|Vol 6|No 4 pp283-298). In brief, the parent (WT), sba1Δ and gcn5Δ yeast were cultured overnight in rich medium (YPD) at 30°C, diluted into 500 mL of fresh YPD to an OD595 = 0.25 and allowed to propagate to OD595 = 1.0. Nuclei were prepared by collecting the cells by centrifugation (5 min at 3000xg), washing the cells 1x with cold dH2O, washing the cells 2x with spheroplasting buffer (50 mM potassium phosphate pH 6.5, 1 M sorbitol, 0.018% ß-mercaptoethanol), resuspending the cell pellets in spheroplasting buffer (4.5 mL) supplemented with Zymolase 20T (40 U mL-1), incubating the cells at 30°C for 45 min with gentle agitation, adding 25 mL lysis buffer (20 mM potassium phosphate pH 6.8, 18% Ficoll 400, 1 mM CaCl2, 0.5 mM EDTA, 3 mM DTT, 1 mM PMSF, 2 mM Benzamidine, 0.5 µg mL-1 Leupeptin and 1.5 µg mL-1 Pepstatin) and lysing the cells with 20 strokes of a Dounce homogenizer using a pestle A.The samples were clarified by centrifugation at 3000xg for 10 min at 4°C, the supernatant was transferred to fresh tubes, samples were clarified by centrifugation at 6700xg for 10 min at 4°C, the supernatant was again transferred to fresh tubes and clarified by centrifugation at 50,000xg for 35 min at 4°C. The supernatants were carefully removed and the nuclei pellets were used for DNase I treatment. To digest the DNA, the yeast nuclei were resuspended in 2.5 mL cold Buffer A (15 mM Tris-HCl pH 8.0, 15 mM NaCl, 60 mM KCl, 1 mM EDTA, 0.5 mM EGTA, 0.5 mM spermidine and 11% sucrose) by gentle swirling and agitation. Nuclei were counted by light microscopy with a typical yield being 2x109 nuclei per preparation. The mixtures were separated into 10 aliquots of 250 µL, each sample was combined with 250 µL 2x reaction buffer (Buffer A supplemented with 12 mM CaCl2,150 mM NaCl and varying levels of DNase I (Roche Applied Science) to give 0, 0.5, 1, 2 or 4 U ml-1). The reactions were incubated at 22°C for 5 min and terminated by the addition of 500 µl stop solution (50 mM Tris-HCl pH 8.0, 100 mM NaCl, 0.1% SDS, 100 mM EDTA, 10 mg ml-1 Ribonuclease A, 1 mM spermidine and 0.3 mM spermine). Pronase was added to 20 mg ml-1 and the reaction was incubated at 55°C overnight. Aliquots (10 µl) of the digested samples were resolved on a 1% agarose gel to validate digestion quality and to select the DNase I concentration at which a very small portion of genomic DNA was digested and a smear under the major high molecular weight band appeared. The 2 U ml-1 DNase I condition was used to proceed to DNA size fractionation and construction of the libraries for deep-sequencing. DNA fragments of 100-500 bp in length were recovered following size fractionation on a 1% agarose gel and purified using microspin columns (Qiagen). DNase I libraries were constructed using the TruSeq DNA Sample Prep kit (Illumina) with the following modifications: 10 ng per sample were used as DNA input, adaptors were diluted 1:20 and final amplification of the adaptored DNA was done for 12 cycles. The libraries were were quantitated by qPCR (Library Quantification Kit, Kapa Biosystems). and equimolar amounts were combined.
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| Library strategy |
DNase-Hypersensitivity |
| Library source |
genomic |
| Library selection |
DNAse |
| Instrument model |
Illumina HiSeq 2000 |
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| Data processing |
The libraries for Dnase-seq were sequenced on 1 lane for 100 cycles with an Illumina HiSeq2000 and TruSeq SBS sequencing kits version 3. Basecalls were performed with Casava1.8 (pipeline 1.9).
The generated sequence data were evaluated and aligned to the yeast reference genome (sacCer3, UCSC) using bowtie (version 0.12.7) with following command: bowtie -p 4 -t --sam --best -v 1 -m 1 ~/indexes/SacCer3. Only uniquely mapped reads were retained for further analyses.
The DNase I hypersensitive regions were identified using the F-seq Peak Caller v1.84 (command:fseq -v -f 0 -of bed -d ./WT -o ./WT
DNA footprints within the DHSs were identified using a described algorithm (NATURE METHODS|Vol 6|No 4 pp283-298)
Genome_build: sacCer3 UCSC
Supplementary_files_format_and_content: Bed files contain the location of DHSs called by fseq (version 1.84). The fifth column is the kernel density at peak summits.
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| Submission date |
Aug 21, 2012 |
| Last update date |
May 15, 2019 |
| Contact name |
Brian Freeman |
| E-mail(s) |
bfreeman@life.illinois.edu
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| Organization name |
UIUC
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| Department |
Department of Molecular & Integrative Physiology
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| Street address |
B107 CLSL 601 S. Goodwin Avenue
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| City |
Urbana |
| State/province |
Illinois |
| ZIP/Postal code |
61801 |
| Country |
USA |
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| Platform ID |
GPL13821 |
| Series (1) |
| GSE40255 |
The p23 molecular chaperone and GCN5 acetylase jointly modulate protein-DNA dynamics and open chromatin status. |
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| Relations |
| SRA |
SRX180133 |
| BioSample |
SAMN02079139 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM989337_BY4741_sba1_deletion_DHS_peak.bed.gz |
36.4 Kb |
(ftp)(http) |
BED |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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