cell type: Primary normal bronchial epithelial cells (NHBEs) treatment: Medium inhibitor pre-treatment: no
Treatment protocol
After reaching confluency, NHBEs were pretreated with either blank medium or CH223191 for 2 hours, followed by treatment with IL4 and House dust mite extract respectively.
Growth protocol
NHBEs were grown to a confluency of 80% and rested for 12h before stimulation.
Extracted molecule
total RNA
Extraction protocol
NHBEs were directly transferred into RNAcell protect reagent (Qiagen) and stored at -80°C until RNA extraction. Total RNA was extracted using RNeasy Mini Kit (Cat.-No. 74104, Qiagen, Hilden, Germany) with on-column DNase digestion (Cat.-No. 79254, DNase-Free DNase Set, Qiagen) for avoiding DNA contaminations (Zissler UM, 2016 Mucosal Immunology). RNA quantification was performed by ultraviolet–visible spectrophotometry (Nanodrop Technologies, Wilmington, DE, USA), for assessment of the RNA integrity by the RNA 6000 Nano Chip Kit with the Agilent 2100 Bioanalyzer (Agilent Technologies, Waldbronn, Germany).
Label
Cy3
Label protocol
Cyanine-3 (Cy3) labeled cRNA was prepared from 50 ng RNA using the One-Color Agilent Low Input Quick Amp Labeling Kit (Agilent) according to the manufacturer's instructions, followed by RNAeasy column purification (QIAGEN, Hilden, GER). Dye incorporation and cRNA yield were checked with the NanoDrop ND-1000 Spectrophotometer.
Hybridization protocol
0.6 ug of Cy3-labelled cRNA (specific activity >6.0 pmol Cy3/ug cRNA) was fragmented at 60°C for 30 minutes in a reaction volume of 25µl containing 1x Agilent fragmentation buffer and 2x Agilent blocking agent following the manufacturers instructions. On completion of the fragmentation reaction, 25µl of 2x Agilent hybridization buffer was added to the fragmentation mixture and hybridized to Agilent Whole Human Genome Oligo Microarrays (G4112A) for 17 hours at 65°C in a rotating Agilent hybridization oven. After hybridization, microarrays were washed 1 minute at room temperature with GE Wash Buffer 1 (Agilent) and 1 minute with 37°C GE Wash buffer 2 (Agilent), then dried immediately by brief centrifugation.
Scan protocol
Slides were scanned immediately after washing on the Agilent DNA Microarray Scanner (G2505B) using one color scan setting for 8x60k array slides (Scan Area 61x21.6 mm, Scan resolution 3µm, Dye channel is set to Green and 20 bit Tiff.
Description
NHBE Medium
Data processing
The scanned images were analyzed with Feature Extraction Software 11.0.1.1 (Agilent) using default parameters (protocol GE1_1100_Jul11 and Grid: 072363_D_F_20150612) to obtain background subtracted and spatially detrended Processed Signal intensities. Features flagged in Feature Extraction as Feature Non-uniform outliers were excluded. Agilent.SingleColor.28004 microarray, Software GeneSpring 14.5; Threshold: 1.0, Logbase: 2; Normalization: Quantile; Baseline Transformation: None; Filtered on Expression (20.0-100.0); Filtered on Flags (Detected, Not Detected); T test unpaired , P<=:0.05, FC>=1.5
