Nucleic acid analysis

For amplification of plasmid constructs, chemically competent bacteria NEB5-alpha (NEB), a derivate of the E. coli strain DH5α, were transformed by means of heat-shock technique according to the manufacturer’s instructions. To this end, a 50 µl batch of bacteria was thawed on ice and incubated with up to 100 ng of pDNA for 30 minutes on ice before being exposed to heat-shock for 30 seconds at 42°C.

After incubation for 5 minutes on ice, bacteria were mixed with SOC complex growth medium (provided by the manufacturer) and incubated for 60 minutes at 37°C and rotation at 250 rpm. Thereafter,

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bacteria (in appropriately diluted suspension) were plated on a pre-warmed LB-agar plate (10 g/l tryptone, 5 g/l yeast extract, 5 g/l NaCl, 15 g/l agar agar), containing 100 µg/ml ampicillin as antibiotic selection marker and incubated overnight at 37°C. Five ml LB-medium (10 g/l tryptone, 5 g/l yeast extract, 5 g/l NaCl), supplemented with 100 µg/ml ampicillin, were then inoculated with cells from a single colony and incubated overnight at 37°C. An aliquot was mixed with an equal volume of 30% (v/v) glycerol in LB-medium and frozen as bacterial glycerol stock at - 80°C. The remaining cells were pelleted by centrifugation for 1 minute at 12,000 x g and directly subjected to plasmid isolation using the GenELute Plasmid Miniprep Kit (Sigma Aldrich) according to the manufacturer's instructions. Briefly, cells were carefully resuspended in resuspension solution, mixed with lysis solution and incubated for 5 minutes. Thereafter, cell debris and contaminants were precipitated by adding neutralization/binding solution and was pelleted by centrifugation for 10 minutes at 12,000 x g. Prior to transferring the lysate, a binding column was equilibrated with colum preparation solution. After loading the sample, the column was centrifuged for 1 minute at 12,000 x g and was then washed with wash solution. Finally, the DNA was eluted with 50 mM Tris pH 8 by centrifugation for 1 minute at 12,000 x g and was stored at -20°C until further use.

2.2.3.2 Spectrophotometric analysis of nucleic acids

To assess the quantity and purity of nucleic acids in solutions, samples were analysed using the micro-volume spectrophotometer NanoDrop2000c (Thermo Fisher Scientific) according to the manufacturers’

instructions. The absorbance at 260 nm was measured and allowed calculation of the concentration of dsDNA and RNA. To guarantee nucleic acid quality for downstream applications, its purity was assessed by calculating the ratio between the absorbance at 280 nm and 260 nm. A value of ~ 2.0 was considered as reference value for ‘pure’ RNA and ~ 1.8 was accepted as ‘pure’ DNA.

2.2.3.3 Agarose gel electrophoresis

Nucleic acids were separated according to size by means of agarose gel electrophoresis. To this end, an agarose gel solution was prepared by solubilising agarose in TAE (40 mM Tris, 1% (v/v) acetic acid, 1 mM EDTA pH 8) by brief boiling. After adding 0.005% (v/v) Midori Green (NIPPON GENETICS), the agarose solution was poured into a horizontal gel chamber and a comb was inserted. Depending on the nucleic acid sizes to be examined, agarose concentration differed between 1% (w/v) for separation of 1 - 10 kb fragments and up to 2% (w/v) for smaller molecules. After polymerization of the gel, samples were mixed with concentrated loading dye, loaded onto the gel and were run at 110 V. Nucleic acids were detected at 260 nm using the 'Universal Hood II' Molecular Imager Gel Doc System (BIO-RAD) and the Quantity One-4.6.8 software.

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In order to analyse the impact of T. gondii on IFN-γ-induced transcriptional regulation in wild type and mutant STAT1-F77A fibroblasts, total RNA was isolated using the GenElute Mammalian Total RNA kit (Sigma Aldrich) according to the manufacturers’ instructions. To digest any contaminating DNA, the isolated RNA samples were further treated with DNase using the Amplification Grade DNase I Kit (Sigma Aldrich).

Briefly, after experimental treatment of cells as specified in the results section, cells were rinsed with PBS and lysed directly in the cell culture vessel. To irreversible denature RNases, the lysis buffer was supplemented with 2-mercaptoethanol at 1:100 (v/v). After incubation for 1 to 2 minutes, the lysate was filtered through a filtration column by centrifuging the samples for 2 minutes at 14,000 x g. The flow-through was then mixed with an equal volume of 70% ethanol, transferred to a silica resin-containing binding column and was centrifuged for 15 seconds at 14,000 x g. The silica column was then washed once with wash solution 1 and twice with wash solution 2. Centrifugations were carried out for 15 seconds at 14,000 x g. Finally, the binding column was additionally centrifuged for 1 minute to remove all ethanol traces. The RNA was eluted with elution solution and directly subjected to DNase I digestion. To this end, 1/10 volume of 10X Reaction buffer and 1/10 volume of DNase I (1 U/µl) were mixed with each sample. After incubation for 15 minutes, 1/10 volume of stop solution was added. The samples were heated for 10 minutes at 70°C and immediately stored at - 80°C or used for downstream applications.

2.2.3.5 Reverse transcription

Messanger (m)RNA was reverse transcribed into cDNA using the Omniscript reverse transcriptase kit (Qiagen) according to the manufacturers’ instructions. In a total reaction volume of 20 µl, up to 2 µg isolated total RNA were mixed with 0.5 mM of each dNTPs, 1 µM oligo-dT primer and 4 U Omniscript reverse transcriptase in RT buffer. The reaction mixture was additionally supplemented with 10 U of RNase inhibitor (Promega). Reverse transcription of the mRNA template was carried out for 90 minutes at 37°C. Newly synthesised cDNA was then stored at – 20°C until further use.

2.2.3.6 DNA sequencing

DNA sequences were verified by the DNA Sanger sequencing method, which was conducted by SeqLab (Sequence Laboratories, Göttingen, Germany). DNA and sequencing primer were provided at amounts and concentrations as recommended by the company.

2.2.3.7 CpG island prediction

In order to predict CpG islands in distinct IFN-γ-responsive promoters, the sequences of the DNA regions -200 to +1 and +1 to +200 upstream or downstream of the transcriptional start site, respectively, were retrieved from the DataBase of Transcriptional Start Sites (Yamashita et al., 2012). CpG-rich regions

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were then predicted in silico with the online tool CpG Island Searcher (Takai and Jones, 2003).

Thresholds for identifying a CpG island were set as region of at least 100 bps, an observed CpG/expected CpG ratio greater than 0.60, and a GC content of more than 50%.