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Sample GSM1201990 Query DataSets for GSM1201990
Status Public on Oct 09, 2013
Title ChIP-seq_WT_H3_II
Sample type SRA
 
Source name ChIP-seq_WT_H3
Organism Schizosaccharomyces pombe
Characteristics genotype/variation: wild type
chip-antibody: anti-H3
chip-antibody vendor: Abcam
chip-antibody cat. #: ab1791
Growth protocol Strains were cultured at 30°C, using Yeast Extract Supplemented (YES) medium
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.
 
Library strategy ChIP-Seq
Library source genomic
Library selection ChIP
Instrument model Illumina HiSeq 2000
 
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
 
Submission date Aug 06, 2013
Last update date May 15, 2019
Contact name Peter J Park
E-mail(s) peter_park@harvard.edu
Phone 617-432-7373
Organization name Harvard Medical School
Department Center for Biomedical Informatics
Street address 10 Shattuck St
City Boston
State/province MA
ZIP/Postal code 02115
Country USA
 
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
Relations
BioSample SAMN02303434
SRA SRX331895

Supplementary file Size Download File type/resource
GSM1201990_ChIP-seq_WT_H3_II_over_WT_Input_II.wig.gz 2.9 Mb (ftp)(http) WIG
SRA Run SelectorHelp
Raw data are available in SRA
Processed data provided as supplementary file

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