cell type: Alveolar epithelial type II cells sorted by FACS gender: male disease state: pulmonary fibrosis
Treatment protocol
Mice with pulmonary fibrosis were treated with either AAV9-Tert or empty vector and were sacrificed 1 week post-virus inoculation. ATII cells were sorted by FACS from mouse lungs of both groups AAV9-Tert and AAV9-empty vector.
Extracted molecule
total RNA
Extraction protocol
ATII cells were sorted by FACS from mouse lungs of both groups AAV9-Tert and AAV9-empty vector. Lungs were extracted and introduced in HBBS buffer with antibiotic and 1% BSA. Separate the lobules of the lung on a dish mince them with a scalpel. Transfer them to a GentleMacs tube with HBBS, antibiotics, 1% BSA, DNAse I (60 units/mL) (Sigma, DN25) and collagenase type I (70 units/mL) (GIBCO, Cat. Number 17100). Then we run the GentleMac program “lung 1”, after the program incubate the sample at 37ºC for 30 minutes and at the end we run the GentleMac program “lung 2”. Cell suspension was filtered through a 40μm stainer and then centrifuge 1200 rpm 5 min. Cells were resuspended in 2 mL ACK Lysis Buffer to lyse red blood cells. Incubate 4 min. at room temperature. We added DMEM without serum to wash, centrifugate and discard supernatant. At the end, resuspend cells in PBS with EDTA (1mM), Hepes (25 mM) and 3% FBS to start staining with LysoTracker as described in commercial protocol (Molecular Probes, LysoTracker Green DND-26, Cat. Num. L7526) and the following antibodies from Pharmingen (BD Biosciences, San Jose CA): PE antimouse CD45, PE antimouse CD31, APC antimouse EpCAM. DAPI (Sigma, St Louis MO) was used to identify dead cells. Data was collected and the defined populations (CD45-CD31-EpCAM+ LysoTracker+ and LysoTracker-) were sorted using an InFlux cell sorted (BD, San Jose CA), we excluded cell aggregates by using pulse processing in the scatter signals and dead cells in the basis of DAPI staining. All data was analyzed using FlowJo software v9.8.5 (Treestar, Ahsland OR). RNA was extracted from post-caval lobe frozen lungs with RNeasy kit following manufacturer instruction (Qiagen, cat. Nº 73504) and RNA integrity analyzed in an Agilent Bioanalyzer. cDNA was synthesised and analyzed on Agilent´s Mouse Genome DNA microarray, following the manufacturer´s instructions. Microarray background subtraction was carried out using normexp method.
Label
Cy3
Label protocol
Amount of nucleic acid labeled: 100ng. Commercial One-Color Microarray-Based Gene Expression Analysis (Low Input Quick Amp Labeling) version 6.7 kit by following manufacturer instructions. Agilent manual G4140-90040 of Sep2014. Amplification: by RNA polymerases. Briefly, MMLV-RT retrotranscription of sample from a T7 promoter primer is followed by a T7 RNA pol catalysed in vitro transcription reaction in the presence of Cy3-CTP fluorophore. Labeled samples are purified with silica-based spin columns.
Hybridization protocol
Microarray: Mouse Gene Expression G3 60K (Agilent microarray design ID 028005, P/N G4852A). Microarray sequences and annotations are listed in sheet '028005_mm9_20121126'. Hybridization chamber type: SureHyb hybridization chamber (Agilent). Quantity of labeled extract used: 600 ng. Volume: 50 µL. Temperature (ºC): 65. Duration: 17 hours.
Scan protocol
Scanned on an G2505C DNA microarray scanner (Agilent). Images were analyzed by Agilent Feature Extraction Software (ver. 11.5), which performed feature quantitation and background subtraction by spatial detrending algorithms.
Data processing
Microarray background subtraction was carried out using normexp method. To normalize the dataset, we performed loess within arrays normalization and quantiles between arrays normalization.
