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| Status |
Public on Jul 27, 2017 |
| Title |
Flag_flag-clr4W31G_SPY5562 |
| Sample type |
SRA |
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| Source name |
Flag_flag-clr4W31G_SPY5562
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| Organism |
Schizosaccharomyces pombe |
| Characteristics |
genotype: flag-clr4W31G
strain: SPY5562
media: YES
antibody: Flag (Sigma F1804)
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
ChIP was performed as previously described (Yu et al., 2014). Conditions for fixation and immunoprecipitation (IP) are briefly described here. 50 ml cultures of cells were grown overnight (or 24 hours when 2.5 ug/ml tetracycline was added) in YES to an optical density (OD) of 2 and fixed as follows. For H3K9me2, H3K9me3, and Chp1 ChIP, cell were fixed with 1% formaldehyde for 15 min. For Swi6 and Flag-protein ChIP, we used a modified dual-crosslinking protocol in which cells were first incubated at 18 °C for two hours, resuspended in 5 ml of room temperature PBS, and fixed with 1.5 mM ethylene glycol bis-succinimidyl succinate (EGS, Thermo Scientific #21565) for 30 min, followed by addition of formaldehyde to 1% final concentration for another 30 min (Zeng et al., 2006). For Swi6 ChIP in Figure S4, the 2 hr incubation at 18 °C was omitted. For each IP, ~ 2 ug of antibody was pre-incubated with 30 ul of Invitrogen Dynabeads Protein A or Protein G: anti-H3K9me2 (Abcam 1220) with Protein A, anti-H3K4me3 (Millipore 04-745) with protein G, anti-H3K36me3 (Abcam 9050) with protein A, anti-H4K16ac (Active Motif 39167) with protein A, anti-Chp1 (Ab18191) with protein A, anti-Flag (Sigma F1804) with protein G, anti-Swi6 (rabbit polyclonal) with protein A, anti-RNA pol II 8WG16 (BioLegend MMS-126R-500) with protein A. For IP with H3K9me3 antibody, Dynabeads M-280 Streptavidin beads were first incubated with 1 ug of anti-H3K9me3 (Diagenode C15500003), followed by blocking with 5 uM biotin. 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 2000 |
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| Description |
barcode sequence: CATTCCAAG
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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 |
Jun 17, 2016 |
| Last update date |
May 15, 2019 |
| Contact name |
Gloria Jih |
| E-mail(s) |
glowjih@gmail.com
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| Organization name |
Harvard Medical School
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| 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 |
GPL13988 |
| Series (1) |
| GSE83495 |
A transcriptionally permissive histone H3 lysine 9 methylation state enables RNAi-mediated heterochromatin assembly |
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| Relations |
| BioSample |
SAMN05263596 |
| SRA |
SRX1853847 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM2204483_63.tdf |
35.9 Mb |
(ftp)(http) |
TDF |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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