Four days-confluent bMEC cells were stimulated with LPS (0.1 µg/ml) and S. aureus JE02 (2.5 ×105 cells/well) or S. aureus SA003 (2.5 ×105 cells/well). The cultures were maintained at 37°C in a humidified atmosphere of 5% CO2 for 12 h and then harvested for RNA extraction.
Growth protocol
Bovine mammary epithelial cell (bMEC) line was originally established by our group (19). bMECs was derived from mammary gland tissue taken from a 200-days pregnant Holstein cows. Cells were maintained in Dulbecco’s modified Eagle medium (DMEM, Gibco, Paisley, Scotland, UK) supplemented with 20% fetal calf serum (FCS; Sigma-Aldrich, Tokyo, Japan), 100 U/ml penicillin and 100 g/ml streptomycin (Gibco 15140122, Life Technologies), transferrin (5 mg/ml) and sodium acetate (5 mM) as a growth medium. The bMEC cells were plated 2.5 ×105 /cm2 in the six-well cell culture plates (BD Falcon, Tokyo, Japan), and incubated at 37°C in a humidified atmosphere of 5% CO2. The cell culture medium was changed at every 24 h.
Extracted molecule
total RNA
Extraction protocol
Total RNA was extracted from the treated and untreated control cells using PureLink RNA Mini Kit (Life Technology INC., USA) along with on-column DNase treatment. RNA integrity, quality and quantity were evaluated with microcapillary electrophoresis (2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA, USA) using the Agilent RNA 6000 Nano Kit (Agilent Technologies, Santa Clara, CA, USA). Samples with RNA integrity number (RIN) greater than 9 were used for microarray study.
Label
Cy3
Label protocol
Cyanine-3 (Cy3) labeled cRNA was prepared from 200 ng RNA using the One-Color Low Input Quick Amp Labeling Kit (Agilent) according to the manufacturer's instructions, followed by RNAeasy mini spin column purification (QIAGEN, Valencia, CA). Dye incorporation and cRNA yield were checked with the NanoDrop ND-1000 Spectrophotometer.
Hybridization protocol
The Gene Expression Hybridization kit (Agilent Technologies) was used for hybridization. In brief, 1650 ng of Cy3-labelled cRNA (specific activity >10.0 pmol Cy3/ug cRNA) was fragmented at 60°C for 30 minutes in a reaction volume of 250 ml containing 1x Agilent fragmentation buffer and 2x Agilent blocking agent following the manufacturers instructions. On completion of the fragmentation reaction, 250 ml of 2x Agilent hybridization buffer was added to the fragmentation mixture and hybridized to Agilent Bovine (V2) Gene Expression Microarray, 4x44K (G2519F) 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 4x44k array slides (Scan Area 61x21.6 mm, Scan resolution 10um, Dye channel is set to Green and Green PMT is set to 100%).
Description
Control
Data processing
The scanned images were analyzed with Feature Extraction Software 10.7.3.1 (Agilent) using default parameters (protocol GE1-v1_91 and Grid: 023647_D_F_20170629) to obtain background subtracted and spatially detrended Processed Signal intensities. Features flagged in Feature Extraction as Feature Non-uniform outliers were excluded.
Transcriptomic Analysis of Inflammatory Responses in Bovine Mammary Epithelial Cells following Stimulation with Escherichia coli and Staphylococcus aureus
