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| Status |
Public on Oct 09, 2013 |
| Title |
ChIP-seq_WT_H2B_II |
| Sample type |
SRA |
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| Source name |
ChIP-seq_WT_H2B
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| Organism |
Schizosaccharomyces pombe |
| Characteristics |
genotype/variation: wild type
chip-antibody: anti-H2B
chip-antibody vendor: Abcam
chip-antibody cat. #: ab1970
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| Growth protocol |
Strains were cultured at 30°C, using Yeast Extract Supplemented (YES) medium
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
100 ml cultures grown to 1-2 x 107 cells/ml were crosslinked (1% formaldehyde, 30 min) and lysed by bead beating. The chromatin fraction was isolated and sheared to 200-500 bp fragments using a Bioruptor sonicator (Diagenode) or Sonicator 3000 (Misonix). Immunoprecipitations (IPs) were performed overnight at 4°C with 1 μg of anti-H3 (ab1791; Abcam), 2 μg of anti-H2B (ab1970; Abcam), 3 μg of anti-H3K36me3 (ab9050; Abcam), 5 μg of anti-H3K4me3 (ab8580; Abcam), 5 μL of anti-Rpb1 (8WG16; Covance), 5 μg of anti-HA (ab9110; Abcam), or 5 μL of anti-Myc (9E10; Santa Cruz Biotechnology). IPs were coupled to 50 μL of protein-G-sepharose beads (GE Healthcare Life Sciences) at 4°C for 4 hours. Beads were washed and eluted; eluate was reverse-crosslinked overnight at 65°C and incubated with proteinase K and glycogen for 2 hours at 37°C. DNA was purified by phenol chloroform extraction and precipitated in ethanol overnight at -20°C.
Between 1-10 ng of ChIP DNA were processed for each ChIP-seq library. DNA was end repaired with T4 DNA Polymerase (Invitrogen), T4 PNK (NEB) and DNA Pol I, Large Klenow fragment (Invitrogen), and an 'A' base was added using Klenow 3' to 5' exo minus (NEB). 1 pmol of barcoded adaptors were ligated on with T4 DNA ligase (Roche) and the products were PCR amplified with Phusion DNA Polymerase (NEB) and size selected by purification on 2% agarose-EX E-gels (Invitrogen) for fragments between 200-600 bp. Library size and concentration was determined by Bioanalyzer or Tapestation analysis (Agilent) and libraries were pooled equimolar amounts with up to 26 in each sequencing lane. Pooled libraries were gel purified twice, followed by column purification on MinElute columns (Qiagen). At least 10 nM were submitted for analysis on the Illumina Hi-Seq at Tufts University Core Facility.
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| Library strategy |
ChIP-Seq |
| Library source |
genomic |
| Library selection |
ChIP |
| Instrument model |
Illumina HiSeq 2000 |
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| Data processing |
ChIP-seq reads were aligned to S. Pombe assembly ASM294v2 using bowtie with ‘-m 10 --best’ options.
The samples were normalized with BEADS. The fragments were extended in the 3’ end direction to be 100 bp long ("beads extend -threePrim <100-read length>”). Spp defined broad enrichment clusters (z=3) were excluded in the BEADS calculation. Downstream ChIP-seq analysis was performed in 10 bp bins as out by BEADS (“beads tagCount -base 10”).
The input samples were normalized to per 1M fragments over nuclear chromosomes.
The IP samples were normalized such that the median fold enrichment over input is set to 1.
Genome_build: ASM294v2
Supplementary_files_format_and_content: wig files at 10bp resolution show fold enrichment for IP samples and normalized tag counts for input samples
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| Submission date |
Aug 06, 2013 |
| Last update date |
May 15, 2019 |
| Contact name |
Peter J Park |
| E-mail(s) |
peter_park@harvard.edu
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| Phone |
617-432-7373
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| Organization name |
Harvard Medical School
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| Department |
Center for Biomedical Informatics
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| Street address |
10 Shattuck St
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| City |
Boston |
| State/province |
MA |
| ZIP/Postal code |
02115 |
| Country |
USA |
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| Platform ID |
GPL13988 |
| Series (2) |
| GSE49574 |
Spt6 regulates intragenic and antisense transcription, nucleosome positioning, and histone modifications genome-wide in fission yeast [ChIP-seq] |
| GSE49575 |
Spt6 regulates intragenic and antisense transcription, nucleosome positioning, and histone modifications genome-wide in fission yeast |
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| Relations |
| BioSample |
SAMN02303439 |
| SRA |
SRX331889 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM1201984_ChIP-seq_WT_H2B_II_over_WT_Input_II.wig.gz |
2.9 Mb |
(ftp)(http) |
WIG |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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