characteristics.text: ERC000011
characteristics.characteristic: ENA-CHECKLIST
characteristics.text: 2018-07-04T17:02:52Z
characteristics.characteristic: ENA-FIRST-PUBLIC
characteristics.text: 2018-04-04T14:14:18Z
characteristics.characteristic: ENA-LAST-UPDATE
characteristics.text: SAMEA1054136
characteristics.characteristic: External Id
characteristics.text: Charles University in Prague Faculty of Science Department of Cell Biology
characteristics.characteristic: INSDC center name
characteristics.text: 2018-07-04T17:02:52Z
characteristics.characteristic: INSDC first public
characteristics.text: 2018-04-04T14:14:18Z
characteristics.characteristic: INSDC last update
characteristics.text: public
characteristics.characteristic: INSDC status
characteristics.text: ERS2359743
characteristics.characteristic: SRA accession
characteristics.text: E-MTAB-6643:Sample 3
characteristics.characteristic: Submitter Id
characteristics.text: 25 to 40
characteristics.unit: year
characteristics.characteristic: age
characteristics.text: ArrayExpress
characteristics.ontologyTerms: http://www.ebi.ac.uk/efo/EFO_0009737
characteristics.characteristic: broker name
characteristics.text: alternatively activated macrophage
characteristics.ontologyTerms: http://purl.obolibrary.org/obo/CL_0000890
characteristics.characteristic: cell type
characteristics.text: human
characteristics.ontologyTerms: http://purl.obolibrary.org/obo/NCBITaxon_9606
characteristics.characteristic: common name
characteristics.text: Protocols: Five million M2 macrophages were cultured in a 500 μl three-dimensional gel made of bovine skin collagen (Nutragen, Advanced BioMatrix, 1.7 or 4.8 mg/ml) in RPMI-1640 with 1% fetal bovine serum and 100 ng/ml M-CSF (Peprotech) for 48 hours in a 24-well plate. Gels from two wells were transferred into one 2 ml tube and homogenized with Tissue Tearor (BioSpec Products) in 600 μl of RNA extraction solution (60% v/v water-saturated phenol, 3.25 M guanidine thiocyanate, 400 mM sodium acetate buffer pH 4.0, 0.4% w/v N-lauroylsarcosine, 160 mM 2-mercaptoethanol) plus 100 μl of 6 M sodium chloride. Primary human macrophages derived from CD14+ subpopulation of peripheral blood mononuclear cells from healthy donors were differentiated and M2-polarized with M-CSF (100 ng/ml) in RPMI-1640 medium supplemented with 10% fetal bovine serum for 5 - 6 days before the experiments. 200 μl of chlorophorm was added to approx. 1 ml of the lysate and the mixture was vortexed vigorously for 10 seconds. After 30-minute centrifugation at 18,000 g at 4°C, the polar upper phase was transferred to a new tube, volume adjusted to 800 μl with RNase-free water, RNA was precipitated with 600 μl of isopropanol at -20°C overnight and recovered by centrifugation at 18,000 g for 30 minutes. The RNA pellet was washed three times with 600 μl of 75% ethanol and air-dried. Next, the RNA was treated with DNAse I to remove possible genomic DNA contamination. To this end, the pellet was directly dissolved in 100 μl of solution containing 4 units of DNAse I (Thermo Fisher Scientific) in manufacturer-provided reaction buffer and incubated at 37°C for 30 minutes. After that, the RNA was re-purified with RNeasy Protect Mini Kit (Qiagen) according to the manufacturer’s instructions and eluted in 50 μl of RNase-free water. Stranded, Illumina HiSeq-compatible library was constructed with ScriptSeq Complete (Human/Mouse/Rat) library preparation kit (Epicentre) according to the manufacturer’s instructions.
characteristics.characteristic: description
characteristics.text: adult
characteristics.characteristic: developmental stage
characteristics.text: normal
characteristics.ontologyTerms: http://purl.obolibrary.org/obo/PATO_0000461
characteristics.characteristic: disease
characteristics.text: low concentration bovine skin collagen; 1.7 milligram per milliliter
characteristics.characteristic: growth condition
characteristics.text: B
characteristics.characteristic: individual
characteristics.text: Homo sapiens
characteristics.characteristic: organism
characteristics.text: Homo sapiens
characteristics.ontologyTerms: http://purl.obolibrary.org/obo/NCBITaxon_9606
characteristics.characteristic: organism
characteristics.text: blood
characteristics.characteristic: organism part
characteristics.text: Homo sapiens
characteristics.characteristic: scientific_name
characteristics.text: female
characteristics.ontologyTerms: http://purl.obolibrary.org/obo/PATO_0000383
characteristics.characteristic: sex
characteristics.text: Sample 3
characteristics.characteristic: title