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| Status |
Public on Jan 21, 2022 |
| Title |
H3K23ac, Log Growth Replicate 1 |
| Sample type |
SRA |
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| Source name |
S. cerevisiae
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| Organism |
Saccharomyces cerevisiae |
| Characteristics |
strain: pJD47
time point: LOG
antibody: Millipore 07-355
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| Treatment protocol |
Proliferating cultures were pelleted and resuspended in autoclaved water containing 10 mM D3-acetate (Sigma 176079) to induce quiescence. After 7 days, quiescent cultures were pelleted and washed to remove any residual D3-acetate, and proliferation was induced by resuspending in liquid media.
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| Growth protocol |
Saccharomyces cerevisiae glycerol stocks were streaked onto 2% agar plates containing yeast nitrogen base (Difco #233520), synthetic complete amino acids mix (Bufferad #S0051) and 2% glucose. Following overnight incubation at 30°C, starter cultures were inoculated with single colonies and incubated overnight at 30°C. Secondary cultures were inoculated at OD600=0.2 and grown at 30°C until they reached OD600=1.
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
Cultures were cross-linked with 1% formaldehyde at RT for 10 min. Reaction was stopped by adding 135 mM glycine. Cultures were then pelleted, washed and resuspended in FA-lysis buffer containing protease/phosphatase inhibitors and 10 mM Na-butyrate. Cells were lysed using a 0.5 mm Zirconia/Silica beads (RPI #9834) in a Mini-Beadbeater high energy cell disruptor (BioSpec) at 4°C, for 6 times 60 seconds. Samples were cooled on ice for 2 min in-between beatings. Subsequently, lysates were sonicated for 20 min in BioRuptor (Diagenode), using high setting and 30 seconds ON/OFF cycles. Cell debris was removed by centrifugation at 10,000 rcf for 15 min at 4°C. Sonication efficiency was confirmed for each sample by running on 0.8% agarose gel. Equal aliquots of sonicated lysates were used per immunoprecipitation reaction with 5 ul of H3K9ac antibody (Active Motif 39317), 5 ul H3K14ac antibody (Active Motif 39697), 5 ul of H3K18ac antibody (Active Motif 39755), 4 ul H3K23ac antibody (Millipore 07-355), 5 ul H3K27ac antibody (Abcam ab4729), 5 ul of H4K12ac antibody (Abcam ab46983), 5 ul H4K16ac antibody (Millipore 07-329), 5 ul Acs2 antibody (Thermo MA5-14810) or 8 ul TAP antibody (Invitrogen CAB1001) preconjugated to Protein G Dynabeads (Life Technologies). Ten percent of the chromatin was saved as input DNA. ChIP reactions were incubated overnight at 4 °C with rotation and washed three times in wash buffer. Immunoprecipitated DNA was eluted from the beads, reversed cross-linked and purified together with the input DNA.
Exactly 20 ng DNA (either ChIP or input) was used to construct sequencing libraries using the NEBNext Ultra II DNA library preparation kit for Illumina (New England Biolabs; NEB). Libraries were multiplexed using NEBNext Multiplex Oligos for Illumina (dual index primers) and single-end sequenced (75 bp) on the NextSeq 550 platform (Illumina) in accordance with the manufacturer’s protocol.
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| Library strategy |
ChIP-Seq |
| Library source |
genomic |
| Library selection |
ChIP |
| Instrument model |
NextSeq 550 |
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| Data processing |
Demultiplexing: chIPseq tags generated with the NextSeq 550 platform were demultiplexed using native applications on BaseSpace.
Alignment: data were aligned to the sacCer3 reference genome using Bowtie2 v2.3.4.1, (parameters --local -X 1000).
Quality Control: aligned tags were filtered for poor alignments using samtools v1.1 (samtools view -q 5 -bS) and merged over NextSeq lanes (samtools merge -n), sorted by tag ID using samtools sort -n, then filtered for PCR duplicates using PICARD (java -jar picard.jar MarkDuplicates REMOVE_DUPLICATES=True ASSUME_SORT_ORDER=queryname).
Track Creation: supplied bigWigs were created for ChIP–seq data by first pooling replicates and generating coverage maps using BEDtools genomeCoverageBed -bg, then adjusting for library size using the RPM (reads per million) coefficient. The input signal was then subtracted from the ChIP signal. The resulting tracks were converted from bedGraph to bigWig files by first sorting by position (bedSort) then using the bedGraphToBigWig utility from the UCSC Genome Browser.
Genome_build: sacCer3
Supplementary_files_format_and_content: The supplied bigWigs show input-subtracted and library size-adjusted tag pile-ups for the four histone acetyl chIPs as well as ACSS2 and TAP-Tagged ACSS2.
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| Submission date |
Nov 23, 2021 |
| Last update date |
Jan 21, 2022 |
| Contact name |
Gregory Donahue |
| Organization name |
The University of Pennsylvania
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| Department |
Cell & Developmental Biology
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| Lab |
Zaret Lab
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| Street address |
3400 Civic Center Blvd, Bldg 421
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| City |
Philadelphia |
| State/province |
PA |
| ZIP/Postal code |
19104 |
| Country |
USA |
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| Platform ID |
GPL26302 |
| Series (2) |
| GSE189445 |
Enzymatic transfer of acetate on histones from lysine reservoir sites to lysine activating sites (ChIP-Seq) |
| GSE189446 |
Enzymatic transfer of acetate on histones from lysine reservoir sites to lysine activating sites |
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| Relations |
| BioSample |
SAMN23413091 |
| SRA |
SRX13211233 |
| Supplementary data files not provided |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data are available on Series record |
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