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| Status |
Public on Jul 17, 2018 |
| Title |
SPY7357 clr4 K455R/K472R_H3K9me2 |
| Sample type |
SRA |
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| Source name |
SPY7357 clr4 K455R/K472R_H3K9me2
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| Organism |
Schizosaccharomyces pombe |
| Characteristics |
media: YEA
antibody: H3K9me2 (Ab1220)
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
ChIP was performed essentially as described (Jih et al., 2017). Briefly, 50 ml of logarithmically growing cells were fixed with 1% formaldehyde for 15 min at room temperature, quenched with 130 mM glycine for 5 min, harvested and washed twice with 1x TBS (50 mM Tris-HCl, pH 7.5, 150 mM NaCl). Cells were resuspended in 500 µl lysis buffer (50 mM Hepes-KOH, pH 7.5, 500 mM NaCl, 1mM EDTA, 1% Triton X-100, 0.1% SDS, and protease inhibitors) in 2 ml screw-cap tubes and lysed by bead beating with 1 ml acid-washed 0.5 mm glass beads with 6 cycles of 30 sec at 5000 rpm on a MagNA Lyser Instrument (Roche). Extracts were sonicated for 3x20 sec at 50% amplitude using a sonicator (Branson Digital Sonifier). For H3K9me2 ChIP, 2 µg of anti-H3K9me2 antibody (Abcam, ab1220) coupled to 30 µl Dynabeads Protein A (Invitrogen, 100-02D) was used for each immunoprecipitation. For H3K9me3 ChIP, 1 µg of anti-H3K9me3 antibody (Diagenode C15500003) was first incubated with 30 µl Dynabeads M-280 Streptavidin beads (Invitrogen, 11206D), followed by blocking with 5 µM biotin, according to the manufacturer's instructions, prior to be used for each immunoprecipitation. Half (250 µl for ChIP-seq) or one fifth (100 µl for ChIP-qPCR) of sheared chromatin lysate was added to the antibody-bead mixture and incubated for 2 hr at 4°C on a rotating device.
Immunoprecipitated DNA was cleaned up using the Qiagen PCR Purification Kit after reversing cross-links, RNaseA, and proteinase K treatment. Immunoprecipitated DNA was eluted from column with the provided elution buffer (50 ul x 2), subjected to additional shearing in a Qsonica water bath sonicator at 20% amplitude for 15 minutes of total shearing time (each cycle is 15” on + 15” off), followed by vacuum centrifugation to reduce the volume to 30 ul. DNA concentration was measured using Qubit dsDNA HS Kit.1 to 10 ng of immunoprecipitated DNA was used for standard Illumina library construction using barcoded adapters and protocol described previously (Wong et al., 2013). Libraries were pooled and sequenced on the Illumina HiSeq2000 or 2500.
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| Library strategy |
ChIP-Seq |
| Library source |
genomic |
| Library selection |
ChIP |
| Instrument model |
Illumina HiSeq 2500 |
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| Description |
BARCODE SEQUENCE: GGTCCTTGA
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| Data processing |
Raw reads were separated by barcode. Barcode sequence were removed from the reads and attached to the identifier. Reads were mapped using Bowtie's default parameters. Mapped reads were normalized to reads per million, tiled with igvtools, and visualized in Integrated Genome Viewer (IGV).
Genome_build: ASM294v2
Supplementary_files_format_and_content: Mapped reads were normalized to reads per million, tiled with igvtools for visualization in Integrated Genome Viewer (IGV).
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| Submission date |
Mar 21, 2018 |
| Last update date |
Jul 20, 2018 |
| Contact name |
Nahid Iglesias |
| E-mail(s) |
nahid.iglesias@duke.edu
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| Phone |
919-613-3385
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| Organization name |
Duke University
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| Department |
Biomedical Engineering
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| Lab |
Charles Gersbach
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| Street address |
101 Science Drive, CIEMAS Rm. 2323
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| City |
Durham |
| State/province |
NC |
| ZIP/Postal code |
27708 |
| Country |
USA |
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| Platform ID |
GPL17225 |
| Series (1) |
| GSE102905 |
Automethylation-induced conformational switch in Clr4 (Suv39h) maintains epigenetic stability |
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| Relations |
| BioSample |
SAMN08767177 |
| SRA |
SRX3828453 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM3058975_86.tdf |
26.0 Mb |
(ftp)(http) |
TDF |
SRA Run Selector |
| Processed data provided as supplementary file |
| Raw data are available in SRA |
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