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Sample GSM1267073 Query DataSets for GSM1267073
Status Public on Dec 26, 2013
Title WT+Ago1OE
Sample type SRA
 
Source name haploid cells
Organism Schizosaccharomyces pombe
Characteristics genotype: h- ade6-M210 leu1-32
strain number: SPY 1491
plasmid: PDM 817
antibody: H3K9me2A (bcam, ab1220)
Growth protocol Cells were grown in minimal media lacking leucine (EMMC-LEU)
Extracted molecule genomic DNA
Extraction 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, pH 7.6,150 mM NaCl), and flash-frozen. Cell pellets 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. Anti-H3K9me2 antibody (Abcam, ab1220) 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, diluted with 250 ul TE, and treated with 50 ug RNase A at 37°C for 1h. 60 ug glycogen and 5 ul proteinase K (Roche) was added and incubation was continued at 55°C for 1h. 22 ul of 10M LiCl was added and the samples were extracted with phenol/chloroform, and EtOH precipitated.
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 the first 6 nt of each read and are listed in a column for each of the raw files. Each barcode (the 7th nt) should be followed by a T.
 
Library strategy ChIP-Seq
Library source genomic
Library selection ChIP
Instrument model Illumina HiSeq 2000
 
Description barcode: ATCACG
Data processing Basecalls performed using CASAVA-1.8.2
Raw reads were separated by barcode and the first 7 nucleotides (6nt barcode and following 'T') 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: GCA_000002945.1
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 Nov 18, 2013
Last update date May 15, 2019
Contact name Ruby Yu
Organization name Harvard University
Department Cell Biology
Lab Danesh Moazed
Street address 240 Longwood Avenue
City Boston
State/province MA
ZIP/Postal code 02139
Country USA
 
Platform ID GPL13988
Series (2)
GSE52444 Effects of RNAi protein overexpression in mutant cells on H3K9 dimethylation in fission yeast cells
GSE52535 Determinants of heterochromatic siRNA biogenesis and function
Relations
BioSample SAMN02404651
SRA SRX378248

Supplementary file Size Download File type/resource
GSM1267073_wt+Ago1OE.ChIP.norm.igv.gz 47.9 Mb (ftp)(http) IGV
SRA Run SelectorHelp
Raw data are available in SRA
Processed data provided as supplementary file

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