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| Status |
Public on Apr 13, 2014 |
| Title |
20090529_1938_B_BMM_LPS_0240_AcH4 |
| Sample type |
SRA |
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| Source name |
BMM_LPS_0240_AcH4
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| Organism |
Mus musculus |
| Characteristics |
strain: C57BL/6
genotype/variation: wild type
age: 8-12 weeks
tissue: primary murine bone marrow macrophage cells (BMMs)
stimuated with: lipopolysaccharide (LPS)
time after lps stimulation: 240 minutes
chip antibody: Acetylated Histone H4
chip antibody vendor: Millipore
chip antibody cat.#: 06-598
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| Growth protocol |
The BMMs were cultured from female C57BL/6 mice (age 8-12 weeks)
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
For ChIP–Seq analysis, formaldehyde-fixed cells were sonicated and processed for immunoprecipitation. In brief, 3 × 107 BMMs were fixed for 10 min in 1% formaldehyde in PBS, washed in PBS, and harvested by scraping in PBS. Cells were lysed by resuspending cell pellets in RIPA buffer (10mM Tris-HCl, pH8.0, 140 mM NaCl, 1% Triton X-100, 0.1% SDS, 1% Deoxcholic acid sodium salt) and drawing the cell suspensions three times through a 30 gauge needle. Chromatin was sheared using a probe sonicator (130 W Ultrasonic Processor with a 3mm tip, 5 × 60s at 30% maximum setting). Sonication quality was checked by gel electrophoresis. Protein concentrations in the extracts were determined (BioRad DC Protein Assay Kit I # 500-111) and sonicated cell extracts containing 0.5 mg protein were incubated with antibodies overnight at 4oC. Immune complexes were recovered by incubation with a 50%-50% mix of magnetic beads coated with Protein A or Protein G (Dynabeads Protein A Invitrogen # 100.02D, Dynabeads Protein G Invitrogen # 100.04D). The magnetic beads were washed with RIPA buffer and the chromatin was eluted with 1% SDS in TE buffer (10 mM Tris-HCl pH8.0, 1 mM EDTA pH8.0) at 65oC for 15 min. Eluted chromatin was reverse-cross-linked by adding 226 mM NaCl and incubating at 65oC for > 5 hr. Then 36 mM Tris–HCl pH8.0, 9 mM EDTA, and 1.5 U of Proteinase K (Fermentas # EO0491) was added to the samples and they were incubated at 42oC for > 1 hr. DNA was purified using phenol/chloroform/isoamyl alcohol (25:24:1, v:v:v) extraction. The purified immunoprecipitated DNA was prepared for sequencing with the Illumina ChIP-Seq Sample Prep Kit and processed according to the manufacturer’s instructions (Illumina Part # 11257047 Rev. A).
A sequencing library for the Illumina Genome Analyzer was derived from the IP using the Illumina reagent kit (see systemsimmunology.org). Single-ended, 36-cycle sequencing was performed on an Illumina Genome Analyzer, and the raw image data were processed using the Illumina Genome Analysis Pipeline Software on a dedicated sequence data processing system (see Genome Analyzer Pipeline Software User Guide, Illumina, San Diego, CA, USA, v0.3).
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| Library strategy |
ChIP-Seq |
| Library source |
genomic |
| Library selection |
ChIP |
| Instrument model |
Illumina Genome Analyzer |
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| Data processing |
Reads were aligned to the mouse genome using eland extended with an ELAND SEED LENGTH value of 25 and an ELAND MAX MATCHES value of 15, and with the 3′-most base excluded. Reads aligned to the same position and strand were counted only once to eliminate duplicates from PCR amplification.
For all ChIP-Seq samples, aligned reads were processed into extended fragments of length 158 bp, the estimated typical insert size in the sequencing library. This estimated size was determined by assaying representative ChIP-Seq samples (after the PCR amplification step) using the Agilent Bioanalyzer to determine the typical fragment size in the sequencing library, and subtracting the combined size of the two Illumina adaptor molecules.
For each sample, the multiscale representation (MSR) was derived as explained in the manuscript.
Genome_build: mm9
Supplementary_files_format_and_content: Enriched segments of the MSR pruned with T=1.05 and R = 0.2. BED format
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| Submission date |
Jan 27, 2014 |
| Last update date |
May 15, 2019 |
| Contact name |
Theo Knijnenburg |
| E-mail(s) |
tknijnen@systemsbiology.org
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| Organization name |
Institute for Systems Biology
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| Street address |
401 Terry Avenue North
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| City |
Seattle |
| State/province |
WA |
| ZIP/Postal code |
98109 |
| Country |
USA |
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| Platform ID |
GPL9185 |
| Series (1) |
| GSE54414 |
Multiscale representation of genomic signals |
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| Relations |
| BioSample |
SAMN02596685 |
| SRA |
SRX450318 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM1314669_20090529_1938_B_BMM_LPS_0240_AcH4.bed.gz |
225.5 Kb |
(ftp)(http) |
BED |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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