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Sample GSM1633570 Query DataSets for GSM1633570
Status Public on Mar 13, 2015
Title HA_2h 2
Sample type SRA
 
Source name old H3 at 2h
Organism Schizosaccharomyces pombe
Characteristics strain: Hu2549
genotype: H3.2-lox-HA-hygR-Lox-T7, cdc25-22 (ts)
chip antibody: HA (abcam ab9110, lot GR98618-1)
treatment: 36°C (5h) + estradiol (2h)
Growth protocol Liquid cultures were grown in YES media shaking at 180 rpm at 25°C.
Extracted molecule genomic DNA
Extraction protocol Immunoprecipitation was done using indicated antibody from chromatin extracts prepared from mid-logarithmic phase cells. Briefly, cells were fixed in 1% formaldehyde for 30 minutes, quenched with 125 mM glycine, and washed twice in PBS. Cells were resuspended in lysis buffer (0.1% SDS, 50 mM Hepes-KOH, 1 % Triton X-100, 0.1 % sodium deoxucholate, 1 mM EDTA and 150 mM NaCl) and lysed with glass beads in a FastPrep homogenizer. The lysate was sonicated and soluble chromatin fragments with an average size of 300 bp were collected. Chromatin was immunoprecipitated with indicated antibody and protein A magnetic beads overnight. The beads were washed.
With the immunoprecipitate still on the beads, the DNA was polished, A-tailed, and ligated to an Illumina sequencing library adaptor. Digestion lambda exonuclease (final concentration 2.5U/reaction) removed nucleotides from 5′ ends of double-stranded DNA until blocked by the formaldehyde induced protein-DNA crosslink. The crosslinks were reversed (4h at 65°C) and DNA was eluted from the beads. The single-stranded DNA was subsequently made double-stranded by primer annealing and extension. A second sequencing adaptor was ligated. Using indexing primers, the fragments were PCR amplified and gel purified (Qiagen MinElute). Samples were quantified on using Qubit (HS dsDNA).
 
Library strategy ChIP-Seq
Library source genomic
Library selection ChIP
Instrument model Illumina HiSeq 2000
 
Description old H3 at 2h
HA_2h.wig
Data processing Read alignment using Bowtie 2 (default parameters)
SAM files loaded into Podbat 0.6.0 (www.podbat.org) with options "first base only", "resolution"=10, "strand-specific" unchecked, MAPQ>30
Normalized to total # reads and (for T7) additionally input amount.
Exported to wig.
Genome_build: ASM294v2
Supplementary_files_format_and_content: wig format, variable step
 
Submission date Mar 13, 2015
Last update date May 15, 2019
Contact name Karl Ekwall
E-mail(s) karl.ekwall@ki.se
Phone +46 8 6089133
Organization name Karolinska Inst
Street address Alfred Nobels Alle 7
City Stockholm
ZIP/Postal code S-141 89
Country Sweden
 
Platform ID GPL13988
Series (2)
GSE66865 A nucleosome turnover map reveals that the stability of histone H4 Lys20 methylation depend on histone recycling in transcribed chromatin [ChIP-exo]
GSE66866 A nucleosome turnover map reveals that the stability of histone H4 Lys20 methylation depend on histone recycling in transcribed chromatin
Relations
SRA SRX878854
BioSample SAMN03351772

Supplementary data files not provided
SRA Run SelectorHelp
Processed data are available on Series record
Raw data are available in SRA

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