organism: Homo sapiens
description: Protocols: Sputum induction and sMac isolation: Sputum induction was performed as previously described (Zissler U. M. et al, Journal of Allergy and Clinical Immunology, 2018). Briefly, patients inhaled increasing concentrations of hypertonic saline solutions (3, 4, 5% V/V) in three consecutive 7-minute inhalation steps. After each step, patients were asked to produce sputum. Samples were kept and processed on ice in the first 60 minutes after sputum induction. Sputum plugs were selected, pooled and homogenized with Dithiothreitol (Sputolysin Reagent, Calbiochem, Merck, Darmstadt, Germany). The homogenate was diluted with PBS and filtrated through a 70 µm cell filter (BD Biosciences, San Diego, CA, USA). The sputum cell pellet was resuspended with PBS. Next, highly purified sputum macrophages (sMac) were obtained as described previously (Frankenberger M. et al, Molecular Medicine, 2011). Briefly, sputum cells were co-incubated with erythrocytes and human monocyte enrichment cocktail (RosetteSep, Stemcell Technologies, Vancouver, Canada) for 20 min at room temperature. A subsequent density gradient centrifugation on LymphoPrep (Alere Technologies AS, Oslo, Norway) generated a mononuclear layer with a purity of >95% sMac. sMac were lysed in RLT Buffer (Qiagen, Hilden, Germany) with 1% β-mercaptoethanol (Carl Roth, Karlsruhe, Germany) and stored at -80 °C until RNA isolation. aMDM generation: Whole venous blood was layered on Polymorphprep (Alere Technologies AS, Oslo, Norway) <2 hours after collection and centrifuged at 500 x g for 35 minutes without brake at room temperature. CD14-positive monocytes were isolated from PBMC and differentiated to aMDM in the presence of GM-CSF (10 ng/mL, Miltenyi Biotec, Bergisch Gladbach, Germany) and TGF-ß (2 ng/mL, Peprotech, Hamburg, Germany) as described previously (Esser J. et al, Journal of Allergy and Clinical Immunology, 2010; Dietz K. et al, Journal of Allergy and Clinical Immunology, 2017). 2x105 aMDM were seeded in 96-wells and cultured for 24 h prior stimulation with the calcium ionophore A23187 (5 µM, Sigma-Aldrich, Merck, Darmstadt, Germany) for 10 min at 37 °C. Cells were washed with PBS, lysed in RLT Buffer (Qiagen, Hilden, Germany) with 1% β-mercaptoethanol (Carl Roth, Karlsruhe, Germany) and stored at -80 °C until RNA isolation. Total RNA was extracted according to the manufacturer's instructions (Quick-RNA MicroPrep Kit, Zymo Research, Irvine, CA, USA). RNA integrity number (RIN) was determined with the Agilent 2100 BioAnalyzer (RNA 6000 Nano Kit, Agilent). For library preparation, 1 μg of RNA was poly(A) selected, fragmented, and reverse transcribed with the Elute, Prime, Fragment Mix (Illumina). A-tailing, adaptor ligation, and library enrichment were performed as described in the TruSeq Stranded mRNA Sample Prep Guide (Illumina). RNA libraries were assessed for quality and quantity with the Agilent 2100 BioAnalyzer and the Quant-iT PicoGreen dsDNA Assay Kit (Life Technologies).
attributes.tag: ENA-FIRST-PUBLIC
attributes.value: 2020-01-03T15:59:20Z
attributes.tag: ENA-LAST-UPDATE
attributes.value: 2019-05-14T16:55:10Z
attributes.tag: External Id
attributes.value: SAMEA5607516
attributes.tag: INSDC center name
attributes.value: Center of Allergy & Environment (ZAUM) Technische Universitat and Helmholtz Zentrum Munchen
attributes.tag: INSDC first public
attributes.value: 2020-01-03T15:59:20Z
attributes.tag: INSDC last update
attributes.value: 2019-05-14T16:55:10Z
attributes.tag: INSDC status
attributes.value: public
attributes.tag: Submitter Id
attributes.value: E-MTAB-7965:MUC7180_S14
attributes.tag: age
attributes.value: 40
attributes.tag: broker name
attributes.value: ArrayExpress
attributes.tag: cell type
attributes.value: alveolar-like monocyte-derived macrophage
attributes.tag: common name
attributes.value: human
attributes.tag: developmental stage
attributes.value: adult
attributes.tag: disease
attributes.value: Aspirin-exacerbated respiratory disease
attributes.tag: individual
attributes.value: 76
attributes.tag: organism part
attributes.value: blood
attributes.tag: sample name
attributes.value: E-MTAB-7965:MUC7180_S14
attributes.tag: scientific_name
attributes.value: Homo sapiens
attributes.tag: sex
attributes.value: female