cell line: Caco-2 cell type: epithelial colorectal adenocarcinoma cell line infection: C. jejuni cas9 deletion strain (delta_Cj1523) timepoint: 60 minutes after infection
Treatment protocol
To investigate the transcriptome of Caco-2 cells infected with C. jejuni strains, cells were seeded in 6-well plates (Greiner Bio-one) at a density of 1.0 x 10[exp5] cells per well. Cells were allowed to grow to confluence for 19 days and infected; 3 biological replicates (3 different wells) were used per C. jejuni strain per time point. In total, in addition to the t=0 time point at which incubation with bacteria or medium-only (the control) was carried out, 5 time points of RNA extraction were used (30, 60, 120, 180, 240 minutes after infection), rationally chosen based on an earlier study (Louwen et al., 2012).
Growth protocol
Human intestinal epithelial cell line Caco-2 (human epithelial colorectal adenocarcinoma cells) were maintained in Dulbecco's modified Eagle's medium (DMEM) (Thermo Fisher Scientific, Bleiswijk, The Netherlands) supplemented with 10% fetal bovine serum (FBS) (Thermo Fisher Scientific), 100 U/ml penicillin, 100 µg/ml streptomycin and 1% non-essential amino acids (NEAA) (Thermo Fisher Scientific). The cells were cultured in a 75-cm2 flask (Greiner Bio-one, Alphen aan den Rijn, The Netherlands) at 37°C and 5% CO2 in a humidified air incubator.
Extracted molecule
total RNA
Extraction protocol
At each assay time point, 1 ml of TRIzol® reagent (Ambion Life Technologies) was added to the appropriate wells and total RNA was extracted from the Caco-2 cells following the manufacturer's protocol. The extracted, air-dried RNA was resuspended in 100 µl MilliQ water and further purified and desalted using Qiagen RNeasy Mini Kit spin columns, following the manufacturer's instructions to make it suitable for microarray hybridizations. RNA quantity and quality were assessed spectrophotometrically via a NanoDrop device (ND-1000, NanoDrop Technologies, Wilmington, DE, USA) and with 6000 Nano chips via a Bioanalyzer 2100 device (Agilent, Santa Clara, CA, USA), respectively.
Label
biotin
Label protocol
The Ambion WT Expression kit (Life Technologies, cat. no. 4411974), in conjunction with the Affymetrix GeneChip WT Terminal Labelling kit (Affymetrix, Santa Clara, CA; cat. no. 900671), was used for the preparation of labelled cDNA from 100ng of total RNA without rRNA reduction.
Hybridization protocol
Labelled samples were hybridised on Affymetrix GeneChip Human Gene 1.1 ST arrays, provided in plate format. Hybridisation, washing and scanning of the array plates were performed on an Affymetrix GeneTitan Instrument, according to the manufacturer's recommendations. Detailed protocols can be found in the Affymetrix WT Terminal Labelling and Hybridisation User Manual (part no. 702808 revision 4), and are also available upon request.
Scan protocol
The Human Gene 1.1 ST array was scanned on an Affymetrix GeneChip 3000 7G scanner. Detailed methods are described in the eukaryotic section of the GeneChip Expression Analysis Technical Manual, Revision 3, from Affymetrix.
Description
After 60 minutes of Caco-2 cell infection with Campylobacter jejuni cas9 deletion strain, Caco-2 cells were incubated with TriZol reagents until lysis was complete, then RNA extraction and purification were performed. RNA was extracted from a single well of a 6-well microtiter plate.
Data processing
Packages from the Bioconductor project (Gentleman et al., 2004) integrated in an in-house developed on-line management and analysis database for multiplatform microarray experiments (Lin et al., J Integr Bioinform. 2011 Jul 21;8(2):160. doi: 10.2390/biecoll-jib-2011-160) were used for analysing the scanned Affymetrix arrays.
