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Sample GSM2174864 Query DataSets for GSM2174864
Status Public on Dec 19, 2016
Title ura4+::priRNA1-KAN leo1∆ + empty vector (spy6419 + pDM829)
Sample type SRA
Source name ura4+::priRNA1-KAN leo1∆ + empty vector (spy6419 + pDM829)
Organism Schizosaccharomyces pombe
Characteristics media: EMM -LEU
antibody: H3K9me2 antibody (Abcam, Ab1220)
Treatment protocol Cells were fixed with 1% formaldehyde for 15 min at room temperature (RT), then quenched with 130 mM glycine for 5 min at RT, harvested by centrifugation, washed twice with TBS (50 mM Tris.HCl pH 7.6, 150 mM NaCl), and flash frozen.
Growth protocol Cells were cultured overnight in EMM-LEU medium at 32°C and harvested at late log phase (OD600 = 1.5).
Extracted molecule genomic DNA
Extraction protocol Cell pellets were resuspended in 600 μl lysis buffer (50 mM HEPES-KOH pH 7.5, 140 mM NaCl, 1 mM EDTA, 1% Triton X-100, 0.1% Na-Deoxycholate, 0.1% SDS, 1 mM PMSF, protease inhibitor tablet (Roche)), and disrupted by bead beating (MagNA Lyser, Roche) for 3x90 sec at 4500 rpm with 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 50% amplitude (Branson Digital Sonifier), the extract was centrifuged (Eppendorf 5415R) for 15 min at 13,000 rpm. The soluble chromatin was then transferred to a fresh tube. H3K9me2 (Abcam, Ab 1220) was preincubated with Dynabeads Protein A, and for each immunoprecipitation, 2 μg antibody bound to 30 μl beads was added to soluble chromatin. Samples were incubated for 2 hours at 4°C with rotation, after which 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 50 μl preheated buffer (50 mM Tris.HCl pH 8.0, 10 mM EDTA, 1% SDS) at 65˚C for 15 min. The eluate was collected, and 75 μl 1XTE/0.67% SDS was added on the beads, the incubation was repeated and both elutions were combined. Immunoprecipitated samples were incubated overnight at 65˚C in the presence of 0 μg RNase A to reverse crosslink and RNase treated the samples, respectively. 5 μl proteinase K (Roche) was added and incubation was continued at 55°C for 1 h. Samples were purified using PCR purification kit (Qiagen).
Libraries for Illumina sequencing were constructed following the protocol detailed in Wong et al, 2013 (Wong, K. H., Jin, Y. and Moqtaderi, Z. 2013. Multiplex Illumina Sequencing Using DNA Barcoding. Current Protocols in Molecular Biology. 101:7.11:7.11.1–7.11.11.), starting with ~10 ng of immunoprecipitated DNA fragments. Each library was generated with custom-made adapters carrying unique 6 nt barcode sequences at the ligating end. Barcoded libraries were mixed and sequenced with Illumina HiSeq 2000.
Library strategy ChIP-Seq
Library source genomic
Library selection ChIP
Instrument model Illumina HiSeq 2000
Description CAGATC
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 with default configurations. Mapped reads were normalized to reads per million and visualized in Integrated Genome Viewer (IGV)
Genome_build: ASM294v2
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.
Submission date May 23, 2016
Last update date May 15, 2019
Contact name Nahid Iglesias
Phone 919-613-3385
Organization name Duke University
Department Biomedical Engineering
Lab Charles Gersbach
Street address 101 Science Drive, CIEMAS Rm. 2323
City Durham
State/province NC
ZIP/Postal code 27708
Country USA
Platform ID GPL13988
Series (2)
GSE81731 Distinct Functions of Argonaute Slicer in siRNA Maturation and Heterochromatin Formation [ChIP-Seq]
GSE81734 Distinct Functions of Argonaute Slicer in siRNA Maturation and Heterochromatin Formation
BioSample SAMN05163197
SRA SRX1792418

Supplementary file Size Download File type/resource
GSM2174864_06.tdf 17.7 Mb (ftp)(http) TDF
SRA Run SelectorHelp
Raw data are available in SRA
Processed data provided as supplementary file

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