In 75 cm2 cell culture flask, HeLa cells (10 million cells) were infected with 50 MOI of FBG for 1 h, 4 h, 48 h and 336 h; each time point in duplicates. Total RNA was purified from each infection assay for microarray-based transcriptome analyses.
Growth protocol
The human cervical carcinoma cell line HeLa S3 (both un-infected and infected by FBG) was cultivated in DMEM medium, and M. hominis FBG was cultivated in arginine-medium.
Extracted molecule
total RNA
Extraction protocol
Nucleic acids were prepared from each 75 cm2 cell culture flask after washing the adherent HeLa cells with 10 ml PBS twice and subsequent lyses in 650 µl RLT Buffer (RNeasy Kit; Qiagen GmbH, Hilden, Germany). 50 µl lysate was used for genomic DNA preparation (Dneasy Blood and Tissue kit, qiagen) and 600 µl lysate for total RNA preparation (RNeasy kit). Before use of the RNA contaminating traces of DNA were digested with DNase I. Total RNA integrity was checked using an Agilent 2100 Bioanalyzer system (Agilent Technologies, Waldbronn, Germany). All samples of this study showed high quality RNA integrity numbers (RIN 9.2 - 9.8). RNA was further analysed by photometric Nanodrop measurements (Thermo Fisher Scientific GmbH, Dreieich, Germany) and quantified by fluorometric Qubit RNA assays (Life Technologies).
Label
Cy3
Label protocol
Synthesis of cDNA and subsequent fluorescent labelling of cRNA was performed on duplicates of each experimental condition according to the manufacturer´s protocol (Agilent One-Color Microarray-Based Exon Analysis/Low Input Quick Amp WT Labelling Kit; Agilent Technologies, Waldbronn, Germany) with some modifications. Briefly, 100 ng of total RNA were converted to cDNA, followed by in vitro transcription and incorporation of Cy3-CTP into the nascent cRNA. To compensate for the prominent AT content of mycoplasma, equal volumes (0.24 µl) of Cy3-UTP (Enzo Life Sciences, Lörrach, Germany) were added to the labelling reactions.
Hybridization protocol
After fragmentation, the maximum amount of labelled cRNA (2.4 µg) was hybridized to Agilent Mycoplasma hominis GE 8x60k Microarrays (OakLabs GmbH, Berlin, Germany/Agilent Technologies, Boeblingen, Germany) for 48 h at 65 °C.
Scan protocol
Microarrays were scanned as described in the manufacturer´s protocol.
Data processing
Signal intensities on 20 bit tiff images were calculated by Feature Extraction (FE, Vers. 11.0.1.1; Agilent Technologies, Waldbronn, Germany) using a custom grid file (061652_D_F_20140129; OakLabs GmbH, Berlin, Germany). After feature extraction and before any normalization, mycoplasma strain specific oligonucleotide signals were extracted using a specific mask file previously defined by genomic DNA hybridization. Data analyses on the strain specific oligo subsets were conducted with GeneSpring GX (Vers. 12.5; Agilent Technologies). Probe signal intensities were quantile normalized across all samples to reduce inter-array variability. Input data pre-processing was concluded by baseline transformation to the median of all samples.
