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| Status |
Public on Nov 26, 2014 |
| Title |
SPY5090+tet_H3K9me3 |
| Sample type |
SRA |
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| Source name |
SPY5090+tet_H3K9me3
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| Organism |
Schizosaccharomyces pombe |
| Characteristics |
strain: SPY5090
antibody: H3K9me3 (Hattori et al, 2013)
media: YEA + 2.5 ug/mL tetracycline
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
Fixed cells were resuspended in 500 ul lysis buffer (50 mM Hepes-KOH, pH 7.5, 500 mM NaCl, 1mM EDTA, 1% Triton X-100, 0.1% SDS, and protease inhibitors) and disrupted by bead beating (Mini Beadbeater, Biospec products) for 6x30 sec with 1 ml 0.5 mm glass beads. Tubes were punctured and the flow-through was collected in a new tube by centrifugation. After sonication for 3x20 sec at 40% amplitude (Branson Digital Sonifier), the extract was centrifuged (Eppendorf 5415R) for 15 min at 13000 rpm. Antibody was pre-incubated with washed Dynabeads Protein A, and for each immunoprecipitation, 2 ug antibody coupled to 30ul beads was added to 400 ul soluble chromatin, and lysis buffer was added for a final volume of 600 ul. Samples were rotated for 2h at 4°C, the beads were collected on magnetic stands, and washed 3 times with 1 ml lysis buffer and once with 1 ml TE, and eluted with 100 ul pre-heated buffer (50 mM Tris pH 8.0, 10mM EDTA, 1% SDS) at 65°C for 15 min. The eluate was collected and 150 ul 1xTE/0.67% SDS was added. Input and immunoprecipitated samples were incubated overnight at 65°C to reverse crosslinks, and treated with 50 ug RNase A at 37°C for 1h. DNA was cleaned up with QIAquick PCR Purification Kit (Qiagen).
Libraries for Illumina sequencing were constructed following the manufacturer’s protocols, starting with ∼5 ng of immune-precipitated DNA fragments. Each library was generated with custom-made adapters carrying unique barcode sequence at the ligating end (Wong and Struhl, 2011). Barcoded libraries were mixed and sequenced with Illumina HiSeq2000. Barcodes are 6/8/9 nt in length and should be followed by a T.
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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 and the barcode sequence were removed from the reads and attached to the identifier. Reads were mapped using bowtie with default configurations. Mapped reads were normalized to reads per million and visualized in Integrated Genome Viewer (IGV)
Genome_build: ASM294v1
Supplementary_files_format_and_content: Mapped reads were normalized to reads per million and visualized in Integrated Genome Viewer (IGV) in an IGV-viewable format.
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| Submission date |
Nov 14, 2014 |
| Last update date |
May 15, 2019 |
| Contact name |
Gloria Jih |
| E-mail(s) |
[email protected]
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| Organization name |
Harvard Medical School
|
| Department |
Cell Biology
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| Lab |
Danesh Moazed
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| Street address |
240 Longwood Ave, LHRRB 517
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| City |
Boston |
| State/province |
MA |
| ZIP/Postal code |
02115 |
| Country |
USA |
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| Platform ID |
GPL17225 |
| Series (1) |
| GSE63304 |
Epigenetic inheritance uncoupled from sequence-specific recruitment |
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| Relations |
| BioSample |
SAMN03195710 |
| SRA |
SRX759788 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM1545356_17.tdf |
36.2 Mb |
(ftp)(http) |
TDF |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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