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| Status |
Public on Sep 26, 2014 |
| Title |
Blueprint_S00NM5H1_H3K27ac |
| Sample type |
SRA |
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| Source name |
Macrophages(BC17)
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| Organism |
Homo sapiens |
| Characteristics |
cell type: Macrophages(BC17)
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| Treatment protocol |
Monocytes were pre-incubated either with cell culture medium (RPMI), β-glucan (5µg/mL) or with LPS (100ng/mL), for 24 hours in a total volume of 10 mL. After a wash-out, cells were cultured in RPMI supplemented with 10% human pool serum. Monocytes were collected at different time points (0 h and 6 d after treatment) and counted before further treatment for chromatin immunoprecipitation or DNaseI treatment.
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| Growth protocol |
Monocytes were pre-incubated either with cell culture medium (RPMI), β-glucan (5µg/mL) or with LPS (100ng/mL), for 24 hours in a total volume of 10 mL. After a wash-out, cells were cultured in RPMI supplemented with 10% human pool serum.
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
Chromatin was fixed in 1% formaldehyde for 10 minutes, sheared to 200-700bp using a Bioruptor (Diagenode), immunoprecipitated using the indicated antisera, purified and sequenced using HiSeq 2000 as recommended by the manufacturer. DNaseI treatment was done on isolated nuclei for 3 minutes and reaction was stopped with stop buffer ( 50mM Tris-HCl, pH 8, 100mM NaCl, 0.10% SDS, 100mM EDTA, pH 8.0, 1mM Spermidine, 0.3mM Spermine), sample was further fractionated on 9% Sucrose gradient for 24hrs/25000rpm at 16ºC . Further sequenced using HiSeq as recommended by the manufacturer.
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| Library strategy |
ChIP-Seq |
| Library source |
genomic |
| Library selection |
ChIP |
| Instrument model |
Illumina NextSeq 500 |
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| Description |
chromatin IP
raw file: H0LRGAGXX_1_5638.sorted.bam
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| Data processing |
Cluster generation and sequencing were performed using the Illumina HiSeq2000 genome analyzer. The image files were processed to extract DNA sequence data using CASAVA-1.8.2. The 42 bp sequence tags were mapped to the reference human genome hg19 (NCBI build37), using the BWA allowing one mismatch. Only uniquely mapped-reads were used for data analysis and visualization. The output data were converted to bam files for downstream analysis. Details about aligner version and parameters used are stored in the headers of the BAM files. PCR duplicates were removed, therefore the final BAM files only contain uniquely mapped reads. Wig files were generated by counting the number of sequence reads (directionally extended to a final 300 bp) per 10-bp genomic window and can be used for viewing the data in the UCSC Genome Browser.
DNAseI bam files were merged per source-treatment (Monocytes RPMI, Macrophages RPMI, Macrophages LPS, Macrophage BG), and DNaseI hotspots were called on the merged bam using the published “Hotspots” pipeline (Neph et al 2012).
Peak calling was performed using MACS2-2.0.10.09132012 using Blueprint C000S5H2 Input Monocytes as a reference input.
For archiving RNA-seq 42-bp reads were aligned to hg19 using GSNAP release 2012-07-20 using parameters -N 1 -n 1 -m 1 -Q.
Gene expression quantification was performed by first aligning RNA-Seq tags to the Ensembl GRCh37 version 68 transcriptome using bowtie with parameters "-a --best --strata -S -m 100 --chunkmbs 256". Name sorted bamfiles were converted using bam2hits and gene expression was quantitated using mmseq (doi: 10.1186/gb-2011-12-2-r13). Results are given in log (base e) RPKM.
Genome_build: hg19
Supplementary_files_format_and_content: Bed.gz files are the peak regions called by MACS2, wig.gz files visualizes the mapped data in the UCSC genome browser. RNA-Seq results are reported as log (base e) RPKM.
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| Submission date |
Jun 09, 2014 |
| Last update date |
May 15, 2019 |
| Contact name |
Hindrik Kerstens |
| Organization name |
Radboud University
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| Department |
Molecular Biology
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| Lab |
Stunnenberg
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| Street address |
Geert Grooteplein Zuid 26
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| City |
Nijmegen |
| ZIP/Postal code |
6525 GA |
| Country |
Netherlands |
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| Platform ID |
GPL18573 |
| Series (1) |
| GSE58310 |
Epigenetic programming during monocyte to macrophage differentiation and trained innate immunity |
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| Relations |
| BioSample |
SAMN02843341 |
| SRA |
SRX581539 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM1406301_LPS_H3K27ac_5638_NR.bed.gz |
126.1 Mb |
(ftp)(http) |
BED |
| GSM1406301_LPS_H3K27ac_5638_NR.wig.gz |
23.7 Mb |
(ftp)(http) |
WIG |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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