characteristics: ERC000011
characteristics: ENA-CHECKLIST
characteristics: 2022-10-03T00:20:53Z
characteristics: ENA-FIRST-PUBLIC
characteristics: 2022-10-03T00:20:53Z
characteristics: ENA-LAST-UPDATE
characteristics: SAMEA110139354
characteristics: External Id
characteristics: Senior Data Scientist
characteristics: INSDC center name
characteristics: 2022-10-03T00:20:53Z
characteristics: INSDC first public
characteristics: 2022-10-03T00:20:53Z
characteristics: INSDC last update
characteristics: public
characteristics: INSDC status
characteristics: ERS12238174
characteristics: SRA accession
characteristics: E-MTAB-11832:NHBECC_T18h_36
characteristics: Submitter Id
characteristics: ArrayExpress
characteristics: broker name
characteristics: NHBE
characteristics: cell line
characteristics: epithelial cell of lung
characteristics: cell type
characteristics: human
characteristics: common name
characteristics: Protocols: Normal human lung fibroblasts (NHLF; Lonza, CC-2512) and IPF patient-derived fibroblasts (IHLF; Asterand, 16769) were used between passage 4 and 8 and cultivated in fibroblast growth medium 2 (FGM-2; Lonza), supplemented with 100 units/ml of penicillin and 100 µg/ml of streptomycin, following the supplier's instructions. Normal human bronchial epithelial cells (i.e. NHBE; Lonza, CC-2541), NHBEC (Lonza, CC-2540) or HBEpiC (ScienCell, 3210-1ea), as well as human tracheal epithelial cells (HTEC; Cell Applications, 06090742) were used between passage 3 and 4 and cultivated in bronchial epithelial cell growth medium (BEGM; Lonza) supplemented according to the supplier’s instructions. The human lung epithelial cell lines NCI-H2452 (ATCC, CRL-5946), A549 (ATCC, CCL-185), NCI-H1975 (ATCC, CRL-5908) and NCI-H1650 (ATCC, CRL-5883) were cultured in RPMI 1640 (Gibco) supplemented with 10% (vol/vol) heat-inactivated FBS (Gibco) and 100 units/ml of penicillin and 100 µg/ml of streptomycin. Human lung epithelial cell lines Calu3 (ATCC, HTB-55) and A427 (ATCC, HTB-53) were cultivated in MEM Eagle medium (Gibco) supplemented with 10% (vol/vol) heat-inactivated FBS (Gibco), 2 mM L-Glutamine, 0.1 mM non-essential amino acids (NEAA), 1 mM Sodium Pyruvate and 100 units/ml of penicillin and 100 µg/ml of streptomycin. NHLF/ NHBE co-culture: NHLF were resuspended in FGM-2 growth medium (Lonza), mixed with an equal volume of NHBE in BEGM growth medium (Lonza), and seeded into a 96-well flat-bottom culture plate (Corning) at a density of 20000 and 1600 cells per well, respectively. Compound dilution series were added, and the co-culture was incubated for 18h at 37°C/5 % CO2. The medium was then replaced with FBM (Lonza) containing 0.1 % faf BSA and the compounds at the indicated concentrations. The medium was supplemented with 100 units/ml penicillin, 100 μg/ml streptomycin for the entire duration of the culture. If not indicated otherwise the co-culture was incubated at 37°C/5 % CO2 for 96 h. Cells were isolated by FACS and lysed in RL Lysis Buffer from NORGEN Single cell RNA purification kit (Norgen). Total RNA was isolated using NORGEN Single cell RNA purification kit including a DNase treatment (Norgen), according to the manufacturer's instructions. Quality control was performed using Qubit 3.0 and RNA HS kit (Thermo Fisher Scientific). Quality was assessed with RNA PicoLabChip (Agilent Technologie) on a Bioanalyzer. In all samples the ratio of absorption at 260 nm/280 nm was between 1.8 and 2.2 u and the RNA Integrity Number (RIN) was > 8.00. All samples passed the quality thresholds. RNA-Seq libraries of poly(A)-selected RNA were isolated from 50 ng of total RNA using NuGen Universal Plus mRNA-Seq kit (NuGen) according to the manufacturer’s recommendations. Final library concentration was determined with Qubit HS cDNA kit (ThermoFisherScientific) and quality was assessed with the Fragment Analyzer HS NGS kit (Agilent). Evaluation of the size, was performed based on a smear range of 100-1000 bp with average size around 330 bp. In parallel qPCR quantification was performed using KAPA library quantification kit (Roche).
characteristics: description
characteristics: pulmonary fibrosis
characteristics: disease
characteristics: wild type genotype
characteristics: genotype
characteristics: co-culture with NHLF
characteristics: growth condition
characteristics: Homo sapiens
characteristics: organism
characteristics: Homo sapiens
characteristics: organism
characteristics: epithelium of bronchus
characteristics: organism part
characteristics: Homo sapiens
characteristics: scientific_name
characteristics: NHBECC_T18h_36
characteristics: title