|
| Status |
Public on Sep 01, 2015 |
| Title |
Donor 3 - Input |
| Sample type |
SRA |
| |
|
| Source name |
Human macrophages differentiated from primary CD14+ monocytes
|
| Organism |
Homo sapiens |
| Characteristics |
cell type: Primary human macrophages
differentiation stage: Day 11
chip antibody: none
|
| Growth protocol |
Primary human monocytes were extracted from the blood samples of three anonymous healthy male donors, donated by the blood transfer centre of the Luxembourgish Red Cross and were used in agreement with the convention between the Luxembourgish Red Cross and the University of Luxembourg from 16.05 2011. Extraction from blood was done by using anti-CD14-antibodies conjugated with superparamagnetic particles {CD14 MicroBeads (Miltenyi Biotec)}, a magnet {MACS separator (Miltenyi Biotec)} and LS Columns (Miltenyi Biotec). After the successful isolation of the CD14+ monocytes, the cells were counted and seeded in a density of 2 million cells/ml on a 10 cm2 plates (about 20 million cells) (Thermo scientific) in order to perform ChIP experiments.For culturing and differentiation of monocytes to macrophages RPMI 1640 medium (VWR) was supplemented with 10% human serum {off the clot, type AB (A&E Scientific, PAA, Pasching, Austria, lot number: C02108-1021)}, 0.1 mg/ml streptomycin (Invitrogen, Life Technologies), 100 U/ml penicillin (Invitrogen, Life Technologies) and 0.1 mM L-glutamine (Invitrogen, Life Technologies). The cells were kept at 37°C under a 5% CO2 atmosphere. The medium was changed, during the differentiation process of monocytes to macrophages, 4 and 7 days after seeding.
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
In order to perform ChIP experiments the chromatin of day 11 cells was cross-linked for 8 min with 1% formaldehyde in PBS. Then the formaldehyde was quenched for 5 min with 125 mM of glycine. Nuclei were isolated and the chromatin was sheared with 30 cycles at high intensity (30 sec off and 30 sec on) using a sonicator (BioruptorTM Next Gene, Diagenode). For each immunoprecipitation 5 μg of sheared chromatin was used. Pre-cleared chromatin was incubated overnight with 5 μg of an antibody against the active enhancer mark H3K27ac (Abcam; ab4729). Following day the antibodies were captured with 25 μl of protein A magnetic beads (Millipore)s for 2 hours on a rotating wheel at 4°C. The DNA was purified with a QIAquick PCR clean-up kit.
ChIP sequencing was performed at the GeneCore facility of EMBL Heidelberg (http://www.genecore.embl.de/) according to the Illumina protocol.
|
| |
|
| Library strategy |
ChIP-Seq |
| Library source |
genomic |
| Library selection |
ChIP |
| Instrument model |
Illumina Genome Analyzer II |
| |
|
| Description |
Input DNA
|
| Data processing |
After sequencing the quality of the raw reads was controlled by FastQC (http://www.bioinformatics.babraham.ac.uk/projects/fastqc/). The reads that had a low quality base pair calling or that were detected as read artefacts were removed (requiring minimum quality score of phred 10 across the read length). Read stacks were collapsed using the FASTX software (http://hannonlab.cshl.edu/fastx_toolkit/index.html).
Reads were aligned with Bowtie v0.1.25 with one mismatch and maximum three locations in the genome allowed (from which the highest quality match was reported).
QuEST v.2.4 was used in order to identify enriched regions. The ChIP enrichment was set to 15 and the ChIP to background enrichment to 3.
Genome_build: hg19
|
| |
|
| Submission date |
May 12, 2015 |
| Last update date |
May 15, 2019 |
| Contact name |
Lasse Sinkkonen |
| E-mail(s) |
lasse.sinkkonen@uni.lu
|
| Organization name |
University of Luxembourg
|
| Department |
Life Sciences Research Unit
|
| Street address |
162a, Avenue de la Faiencerie
|
| City |
Luxembourg |
| ZIP/Postal code |
1511 |
| Country |
Luxembourg |
| |
|
| Platform ID |
GPL9115 |
| Series (1) |
| GSE68798 |
Integrated metabolic modelling reveals cell-type specific epigenetic control points of the macrophage metabolic network |
|
| Relations |
| SRA |
SRX1023939 |
| BioSample |
SAMN03656345 |
