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| Status |
Public on Jul 10, 2014 |
| Title |
U2OS_H3K4me3_ChIPseq |
| Sample type |
SRA |
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| Source name |
U2OS cells
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| Organism |
Homo sapiens |
| Characteristics |
antibody: Histone H3 tri-methyl K4( Abcam, ab8580)
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| Treatment protocol |
U2OS cells were induced with doxycycline (1µg/ml; shMiz1: 0.05µg/ml) for 30h. Acute deletion of Miz1∆POZ was achieved with 200nM 4-OHT.
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| Growth protocol |
HeLa, U2OS and pancreas cells were grown in DMEM (Sigma) supplemented with 10% fetal calf serum (Biochrom) and penicillin/streptomycin (Sigma). MEFs were grown in DMEM (Sigma) supplemented with 10% fetal calf serum (Biochrom), 1x non-essential amino acids (Sigma), 1% penicillin/streptamycin (Sigma) and 50µM beta-mercaptoethanol. T cells were grown in RPMI (Sigma) supplemented with 10% fetal calf serum (Biochrom), 1x non-essential amino acids (Sigma), 1% penicillin/streptamycin (Sigma) and 50µM beta-mercaptoethanol.
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
For ChIP-seq cells were crosslinked with 1 % formaldehyde for 10' at 37°C. After cell lysis nuclei were resuspended in RIPA buffer and sonicated (Bransson) until average fragment size was below 500 bps. Antibodies were bound to Protein-A/G-sepharose or Dyna beads and incubated with the chromatin. After sequential washing and elution with 1 % SDS, crosslinking was reverted and DNA was purified using Qiagen PCR purification columns.
Libraries were contructed following manufactor's intructions (NEBNext ChIP-Seq Sample Prep Kit). Briefly, ChIP DNA was end repaired, A tailed and Illumina adaptors were ligated. DNA fragments of about 200 bps were cut out of a agarose gel and extracted with a Qiagen PCR purification column. Afterwards, DNA was enriched with 18 PCR cycles, fragment size was controlled with Biorad Experion system an quantified using picogreen assay. Libraries were sequenced on an Illumina Genome Analyzer IIx following the manufactor's instructions.
For RNAseq, poly-A RNA was isolated from total with Sera-Mag Oligo(dT) Magnetic Particles (Thermo Scientific).
Library preparation was performed by using the NEBnext® mRNA Library Prep Master Mix set for Illumina (E6100S/L) following the instruction manual. Briefly, poly-A RNA was frangmented to generate 200 nucleotides fragments. First and second strand synthesis was performed and the resulting cDNA was end-repaired, ligated to NEBnext Adaptor, size selected (250bps) and purified with Qiagen gel extraction kit. cDNA was then amplified with 12-15 cycle of PCR and the resulting library was subjected to Illumina GAIIx sequencing according to the manufacturer instructions.
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| Library strategy |
ChIP-Seq |
| Library source |
genomic |
| Library selection |
ChIP |
| Instrument model |
Illumina Genome Analyzer IIx |
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| Data processing |
Basecalling was performed with the RTA package within the Genome Analyzer Data Collection Software (SCS2.8).
Fastq files were generated by CASAVA only considering high quality sequences (PF-cluster).
Overall sequencing quality was checked with the FastQC script.
Reads were aligned to the human genome with BOWTIE v0.12.7 or v0.12.8 using default parameters.
For ChIPseqs peak calling was performed with MACS v1.4.2 with default parameters, but depending on the sample: model fold 15 to 25, duplicates 1 to 100, p-value of 1E-5 to 1E-6 and FDR 0.1
For ChIPseqs of histone modifications SICER v1.1 with the following parameters has been used: redundancy 1, window size 200, gap size 200, fragment size 150, FDR 5E-2 to 1E-4.
For RNAseqs reads were aligned to the reference genome using BOWTIE v0.12.7 or v0.12.8
Differential expression was identified using EdgeR. For RNAseqs in MEFs reads per gene were counted.
Genome_build: hg19 for human and mm9 for murine datasets
Supplementary_files_format_and_content: txt files from ChIP-seq experiments containing peak localisation in a MACS output -bed format. txt files from ChIPseq experiments of histone modifications contain peak localisation in SICER output format. txt files from RNA-seq experiments contain ensembl-ID, gene symbol, log2 fold change, and p(adjust).
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| Submission date |
Mar 24, 2014 |
| Last update date |
May 15, 2019 |
| Contact name |
Martin Eilers |
| Organization name |
University of Wuerzburg
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| Department |
Chair for Biochemistry and Molecular Biology
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| Lab |
Martin Eilers
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| Street address |
Am Hubland
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| City |
Wuerzburg |
| ZIP/Postal code |
97074 |
| Country |
Germany |
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| Platform ID |
GPL10999 |
| Series (1) |
| GSE44672 |
Activation and repression by oncogenic Myc shapes tumour-specific gene expression profiles |
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| Relations |
| BioSample |
SAMN02699370 |
| SRA |
SRX500800 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM1356566_U2OS_H3K4me3.txt.gz |
664.0 Kb |
(ftp)(http) |
TXT |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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