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| Status |
Public on Aug 31, 2014 |
| Title |
Gcn5_ChIP-seq_t9 |
| Sample type |
SRA |
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| Source name |
YMC cycling cells
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| Organism |
Saccharomyces cerevisiae |
| Characteristics |
strain: ZKY428: CEN.PK MATa GCN5-FLAG::natMX, SET1-3HA::KanMX14
time: t9
chip antibody: FLAG M2 [F1804 (Sigma)]
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
~50 OD cycling cells per time point were collected for ChIP 3 µg primary antibody was used per ChIP experiment. Briefly, cells were first fixed in 1% formaldehyde at 25℃ for 15 min and quenched in 125mM glycine at 25℃ for 10 min. Cells were pelleted and washed twice with buffer containing 100 mM NaCl, 10 mM Tris-Cl pH 8.0, 1 mM EDTA, 1 mM PMSF, 1 mM benzamidine•HCl before freezing. The frozen pellet was resuspended 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 (DOC), 0.1% SDS, 1 mM PMSF, 10 µM leupeptin, 5 µM pepstatin A, Roche protease inhibitor cocktail) and lysed by bead beating. Lysate from 50 OD cells were split into two tubes each containing 280 µl lysate and sonicated for 16 cycles (30 sec on, 1 min off, high output) using a Bioruptor (Diagenode, Denville, NJ or Tosho Denki, Japan). The supernatant of the sonicated lysate was pre-cleared. 50 µl lysate was saved as input. For ChIP with histone modifier proteins, 500 µl whole cell extract (WCE) was diluted 1:10 and used for each ChIP. After incubation overnight, 50 µl protein G magnetic beads (Invitrogen, Grand Island, NY, 10003D) resuspended in ChIP lysis buffer was added and incubated for 1.5 h at 4 ℃. Beads were washed twice with ChIP lysis buffer, twice with DOC buffer (10 mM Tris•Cl pH 8.0, 0.25 M LiCl, 0.5% deoxycholate, 0.5% NP-40, 1 mM EDTA) and twice with TE. 125 µl of TES buffer (TE pH8.0 with 1% SDS, 150 mM NaCl, and 5 mM dithiothreitol) was added to resuspend the beads. Supernatant was collected after incubation at 65°C for 10 min. A second round of elution was performed and the eluates were combined. Reverse crosslinking was performed by incubation for 6 h at 65°C. An equal volume of TE containing 1.25 mg/ml proteinase K and 0.4 mg/ml glycogen was added to the samples after reverse crosslinking and samples were incubated for 2 h at 37°C. Samples were extracted twice with an equal volume of phenol and once with 25:1 chloroform:isoamyl alcohol. DNA was precipitated in 0.1 volume 3.0 M sodium acetate (PH 5.3) and 2.5 volume of 100% ice-cold ethanol at -20℃ overnight. Pellets were washed once with cold 70% ethanol and resuspended in 20 µl TE.
Library construction and sequencing were performed following the Illumina protocol. Briefly, DNA was end repaired and A-tailed. Barcoded adaptors were ligated and DNA was run in 2% agarose gel. DNA fragments from 150 bp to 300 bp long were excised from gel and used for PCR. PCR products were gel-extracted again and quantified on an Agilent Bioanalyzer.
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| Library strategy |
ChIP-Seq |
| Library source |
genomic |
| Library selection |
ChIP |
| Instrument model |
Illumina HiSeq 2000 |
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| Description |
Same time points as other histone modifiers ChIP-Seq.
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| Data processing |
Sequence reads were mapped to the Saccharomyces.cerevisiae (sacCer2) using bowtie. In order to adjust sequencing depth difference, signals at each window were normalized by total number of mappable reads across 14 time points.
supplementary_files_format_and_content: .txt files show the sequencing read intensity across the sacCer2 genome in CisGenome browser. The first column in the txt file is the name of the chromosome. The second column is the start position of each of the continuous 100 bp windows in the chromosome. The third column is the intensity of reads in this window.
Genome_build: sacCer2
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| Submission date |
Nov 13, 2013 |
| Last update date |
May 15, 2019 |
| Contact name |
Zheng Kuang |
| Organization name |
UT Southwestern medical center
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| Department |
Immunology
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| Lab |
Lora Hooper
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| Street address |
6000 Harry Hines Blvd. NA7.500
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| City |
Dallas |
| State/province |
TX |
| ZIP/Postal code |
75390 |
| Country |
USA |
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| Platform ID |
GPL13821 |
| Series (1) |
| GSE52339 |
A high-resolution view of transcription and chromatin states across distinct metabolic states in budding yeast |
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| Relations |
| BioSample |
SAMN02402161 |
| SRA |
SRX377154 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM1263507_gcn5_9IIBpeakN.txt.gz |
474.9 Kb |
(ftp)(http) |
TXT |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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