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| Status |
Public on Mar 19, 2019 |
| Title |
PU.1_ChIPseq_Ca_stimulated_rep1 |
| Sample type |
SRA |
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| Source name |
HoxER-PU.1 WT neutrophils
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| Organism |
Mus musculus |
| Characteristics |
cell type: HoxER-PU.1 WT neutrophils derived from immortalized progenitors
strain: MRP8-Cre-ires/GFP (PU.1 WT)
chip antibody: PU.1 (Santa Cruz, sc-352X)
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| Treatment protocol |
HoxER-PU.1 WT neutrophils were left unstimulated or were stimulated for 1 hour with Candida albicans heat-inactivated (15 min at 80°C) cell material in an multiplicity of infection of 1.
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| Growth protocol |
Progenitor cells were enriched from BM single cell suspensions of PU.1 WT mice via density-gradient (Lymphocyte Separation Medium, Lonza) centrifugation (400g, 15 min, RT). Progenitors were prestimulated in RPMI (PAA) supplemented with 10 % FCS, 100 μg/ml Penicillin/Streptomycin, 2 mM L-glutamine and 10 ng/ml murine IL-3 (PeproTech), 20 ng/ml murine Il-6 (PeproTech) and 25 ng/ml murine SCF (PeproTech). Progenitor cells were immortalized by spin infection (4000 rpm, 1 h, RT) with a retrovirus expressing Hoxb8 fused to the estrogen receptor (HoxER) and by maintaining the cells in Opti-MEM® (Life Technologies) supplemented with 10 % FCS, 100 μg/ml Penicillin/Streptomycin, 10 ng/ml SCF, 30 µM 2-Mercaptoethanol and 2 µM β-Estradiol (Sigma). Differentiation into neutrophils was induced by withdrawal of Estradiol and addition of murine G-CSF (PeproTech) for 4 days.
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
2x107 HoxER-PU.1 WT neutrophils were harvested in a 50 ml Falcon tube. Cells were crosslinked with 1 % formaldehyde for 10 min at RT while shaking (seesaw rocker SSL4, Stuart). After crosslinking 5 ml 1 M HEPES (pH 7.5) was added and reaction was quenched by addition of 0.125 M glycine (5 min, RT, shaking). Cells were pelleted (1300 rpm, 8 min, 4°C) and washed two times with ice-cold PBS. Cell lysis was performed by swelling the pellet in ice-cold lysis buffer (10 mM Tris, pH 8.0, 10 mM NaCl, 0.2 % NP-40, 1:500 proteinase inhibitor cocktail (PIC, Sigma)) for 10 min on ice. Nuclei were pelleted by centrifugation (1800 rpm, 5 min, 4°C), and were dissolved in Non-SDS sonication buffer (10 mM Tris, pH 8.0, 1 mM EDTA, 0.5 M EGTA, 1:500 PIC). After incubation for 10 min on ice, chromatin was transferred to TPX hard plastic reaction tubes (1.5 ml or 15 ml, Diagenode) and was sonicated for 30 minutes sonication time on high power (30 sec on, 30 sec off, 0.5 sec pulse rate) using a Bioruptor (Diagenode) and a minichiller (Huber) for cooling. After sonication, chromatin was centrifuged (13200 rpm, 15 min, 4°C) to pellet cell debris and an aliquot (10% Input) was removed and was stored at -20°C for later usage. The chromatin was diluted 10x with ChIP dilution buffer (0.01 % SDS, 1.1 % Triton-X-100, 1.2 mM EDTA, 16.7 mM Tris-HCl, pH 8.1, 167 mM NaCl) in a 2 ml reaction tube. To preclear the chromatin, 40 μl Protein A/G Agarose beads (Santa Cruz) were added, and the mixture was incubated for 30 min at 4°C under constant rotation (Rotator SB3, Stuart). Beads were pelleted (1000 rpm, 1min, 4°C) and the supernatant was transferred to a new reaction tube. 10 µg PU.1 antibody (Santa Cruz, sc-352X) or normal rabbit IgG (Upstate, 12-370) were added to the chromatin, and the mixtures were incubated overnight at 4°C under rotation. Protein A/G agarose beads (Santa Cruz) were blocked for one hour at RT in 1ml PBS containing 1.5% fish skin gelatin (Sigma). After washing, beads were resuspended in the initial volume of ChIP dilution buffer. For precipitation, 200 µl protein A/G agarose beads were added to the chromatin/antibody mix, and the solution was incubated for one hour at 4°C under rotation. The beads were pelleted (1000 rpm, 1 min, 4°C) and were washed two times in 1 ml ChIP low salt immune complex wash buffer (0.1 % SDS, 1 % Triton-X-100, 2 mM EDTA, 20 mM Tris-HCl, pH 8.1, 150 mM NaCl) while rotation for 5 min at 4°. Then, beads were washed two times in 1 ml ChIP high salt immune complex wash buffer (0.1 % SDS, 1 % Triton-X-100, 2 mM EDTA, 20 mM Tris-HCl pH 8.1, 500 mM NaCl) and two times in 1 ml ChIP LiCl immune complex wash buffer (0.25 M LiCl, 1 % NP-40, 1 % DOC, 1 mM EDTA, 10 mM Tris, pH 8.1). Afterwards, pelleted beads were washed two times in 1 ml TE buffer (10 mM Tris-HCl, pH 8.0, 1 mM EDTA, pH 8.0). DNA elution was performed by addition of 250 μl ChIP elution buffer (1 % SDS, 0.1 M NaHCO3) and incubation for 15 min at RT under rotation. The beads were pelleted (1000 rpm, 1 min, 4°C) and the supernatant was transferred to a new 1.5 ml Safe-Lock tube. The beads were conducted to a second elution step, and the two eluates were combined. Protein-DNA crosslinks of samples and inputs (10 %) were reversed by addition of 20 μl 5 M NaCl (overnight, 65°C). Next day, 2 μl (20 mg/ml) proteinase K (Invitrogen), 10 µl 0.5M EDTA and 20 µl Tris-HCl pH 6.5 were added and incubated 1h at 45°C. DNA was isolated by addition of one volume phenol/chloroform/isoamyl alcohol (Roth, 25:24:1) and subsequently centrifugation of the mixture (13200 rpm, 15 minutes, 4°C). The aqueous phase containing the DNA was transferred to a new tube, and 1 µl glycogen (20 mg/ml, Sigma) was added. The precipitated DNA was resuspended in 60 µl DNase and RNase free water (Gibco) and was stored at -20°C for further applications.
Libraries were prepared using the TruSeq ChIP Sample Preparation Kit (Set A, Illumina # IP-202-1012) following the instruction of Illumina. PU.1 ChIP-seq libraries were prepared using the TruSeq ChIP Sample Preparation Kit (Set A, Illumina) following the instruction of the manufacturer. Briefly, 5- 50 ng ChIP DNA was used as a start material. As a first step, overhangs resulting from the fragmentation were converted into blunt ends utilising the End Repair Mix. Next, the 3’ ends were adenylated, and indexing adaptors were ligated to the ends of the DNA fragments. For pooling three libraries in 1 lane, Index Adaptors AR002, AR007 and AR019 or AR005, AR006 and AR015 were used. Subsequently, the ligation products were gel-purified, and fragments were size-selected (250-300 bp). Library DNA was amplified by PCR, and the run was reduced to 12 cycles. DNA fragments were purified via bead selection, and ChIP-seq libraries were qualified on an Agilent Technologies 2100 Bioanalyzer by using the High Sensitivity DNA chip. The Core Facility Genomic Muenster performed library quantification by qPCR (Kapa Library Quant Kit) and single-end sequencing on an Illumina HighScanSQ.
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| Library strategy |
ChIP-Seq |
| Library source |
genomic |
| Library selection |
ChIP |
| Instrument model |
Illumina HiScanSQ |
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| Data processing |
Basecalls were performed using Real-Time Analysis (RTA) version 1.13.48.
ChIP-Seq reads were aligned to the current mouse reference sequence (NCBI build 38, mm10) using bowtie2 version 2.0.2 and only uniquely mapped reads were used for downstream analysis.
ChIP-Seq replicates with correlation values ≥0.9 as determined by DiffBind version 2.4.8 were merged and further analysed as one sample.
Peaks were called using MACS version 1.4 with a stringent p-value of 10-10.
Genome_build: mm10
Supplementary_files_format_and_content: Processed data files were created with MACS 1.4 and represent ChIP-seq peaks, including the MACS-specific score per identified peak region.
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| Submission date |
Feb 20, 2018 |
| Last update date |
Oct 28, 2021 |
| Contact name |
Frank Rosenbauer |
| E-mail(s) |
frank.rosenbauer@ukmuenster.de
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| Organization name |
University Hospital Münster
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| Department |
Institute of Molecular Tumor Biology
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| Street address |
Robert-Koch-Str. 43
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| City |
Münster |
| ZIP/Postal code |
48149 |
| Country |
Germany |
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| Platform ID |
GPL16173 |
| Series (2) |
| GSE110863 |
Genome-wide binding profiles of PU.1 in unstimulated and Candida albicans stimulated HoxER-PU.1 WT neutrophils |
| GSE110865 |
Safeguard function of PU.1 shapes the inflammatory epigenome of neutrophils |
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| Relations |
| BioSample |
SAMN08567893 |
| SRA |
SRX3723677 |
| Supplementary data files not provided |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data are available on Series record |
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