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| Status |
Public on Mar 13, 2015 |
| Title |
T7_0h 2 |
| Sample type |
SRA |
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| Source name |
new H3 at 0h
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| Organism |
Schizosaccharomyces pombe |
| Characteristics |
strain: Hu2549
genotype: H3.2-lox-HA-hygR-Lox-T7, cdc25-22 (ts)
chip antibody: T7 (EMD chemicals 69522, lot D00137263
treatment: 36°C (3h)
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| Growth protocol |
Liquid cultures were grown in YES media shaking at 180 rpm at 25°C.
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| 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).
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| Library strategy |
ChIP-Seq |
| Library source |
genomic |
| Library selection |
ChIP |
| Instrument model |
Illumina HiSeq 2000 |
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| Description |
new H3 at 0h
T7_0h.wig
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| 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
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| Submission date |
Mar 13, 2015 |
| Last update date |
May 15, 2019 |
| Contact name |
Karl Ekwall |
| E-mail(s) |
karl.ekwall@ki.se
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| Phone |
+46 8 6089133
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| Organization name |
Karolinska Inst
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| Street address |
Alfred Nobels Alle 7
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| City |
Stockholm |
| ZIP/Postal code |
S-141 89 |
| Country |
Sweden |
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| 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 |
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| Relations |
| SRA |
SRX878819 |
| BioSample |
SAMN03351772 |
| Supplementary data files not provided |
SRA Run Selector |
| Processed data are available on Series record |
| Raw data are available in SRA |
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