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| Status |
Public on Aug 23, 2018 |
| Title |
H3K18ac_t7_ChIPSeq |
| Sample type |
SRA |
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| Source name |
Yeast Metabolic Cycle
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| Organism |
Saccharomyces cerevisiae |
| Characteristics |
strain: ZKY329: CEN.PK MATa SPT7-FLAG::natMX
chip antibody: H3K18ac (Millipore, Mi 07-354)
time: 49.32 hr
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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 antibodies, 50 µl whole cell extract (WCE) was diluted 1:10 and used for each ChIP. After incubation overnight, 50 µl protein A Sepharose beads (GE Healthcare Life Sciences, Piscataway, NJ, 17-5280-01) 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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| Data processing |
Quality filtering was performed using Trimmomatic v0.32
Genome mapping was done using RNA-STAR sofware using S. cerevisiae as reference genome, EnsEMBL release 91 was the version used.
Alignments were filtered for multimapped reads and subject for duplicate removal using Samtools software
Genomecov software (from bedtools) was used for coverage per nucleotide quantification
Genome_build: S. cerevisiae EnsEMBL (release 91)
Supplementary_files_format_and_content: Processed data files are per-base genome coverage. Three columns represent: chromosome, base and number of reads that fall in the given base.
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| Submission date |
Aug 22, 2018 |
| Last update date |
Aug 23, 2018 |
| Contact name |
Salvador Casani |
| E-mail(s) |
salvacasani@gmail.com
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| Organization name |
Dana Farber Cancer Institute
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| Department |
Cancer Biology
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| Lab |
Bernstein Lab
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| Street address |
450 Brookline Avenue
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| City |
Boston |
| State/province |
MA |
| ZIP/Postal code |
02215 |
| Country |
USA |
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| Platform ID |
GPL13821 |
| Series (1) |
| GSE118889 |
Elucidating the regulation of the Yeast Metabolic Cycle through the integration of gene expression and chromatin status |
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| Relations |
| BioSample |
SAMN09874356 |
| SRA |
SRX4590908 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM3349816_H3K18ac-7_ATCACG_L008_R1_001_Coverage.txt.gz |
30.8 Mb |
(ftp)(http) |
TXT |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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