genomewide characterization of host-pathogen interactions by ...
genomewide characterization of host-pathogen interactions by ... genomewide characterization of host-pathogen interactions by ...
Maren Depke Material and Methods Pathogen Gene Expression Profiling Bacterial control samples Bacterial samples were taken as comparison and to control for additional experimental effects (Fig. M.5.2 and Fig. M.5.3): exponential growth phase at OD 0.4 staphylococci after 1 h, 2.5 h, and 6.5 h of incubation in the infection medium in 5 % CO 2 -atmosphere at 37°C without agitation (i.e. in cell culture dishes in serum containing medium but without presence of host cells) non-adherent staphylococci after 1 h of co-incubation with eukaryotic cells and their products in 5 % CO 2 -atmosphere and at 37°C staphylococci after 2.5 h of anaerobic incubation in pMEM medium at 37°C Cells were harvested using Killing Buffer (20 mM Tris pH 7.5, 5 mM MgCl 2 , 20 mM NaN 3 ) as described above (see Material and Methods/Growth Media Comparison Study/Bacterial cell harvest, page 55). exponential growth phase anaerobic incubation: 2.5 h infection mix serum control with CO 2 exposure: 1 h, 2.5 h, 6.5 h non-adherent staphylococci: 1 h Fig. M.5.3: Visualization of bacterial control samples in the in vitro infection experiments for tiling array analysis. internalized staphylococci: 2.5 h, 6.5 h S9 cells staphylococci Preparation of internalized staphylococci The lysostaphin-containing medium was replaced by 1 ml Killing Buffer (20 mM Tris pH 7.5, 5 mM MgCl 2 , 20 mM NaN 3 ) with 150 mM NaCl. In this isotonic buffer the infected S9 cells were scraped from the culture dish, resuspended and transferred to a 1.5-ml tube for further processing while maintaining the integrity of the majority of host cells. All following steps were performed on ice or at 4°C. Cells were pelleted (600 x g for 5 min) and fixed in ice-cold acetone/ethanol (50 % v/v) for 4 min as described by Garzoni et al. (2007) followed by a centrifugation step at 20000 x g for 3 min. The eukaryotic part of the cell pellet was lysed in RLT buffer (Qiagen, Hilden, Germany) and homogenized twice using QIAshredder (Qiagen, Hilden, Germany) and centrifugation at 20000 x g for 2 min. In this process staphylococci were not lysed although they lost their viability. Therefore, the resulting pellet contained staphylococcal cells and eukaryotic cell debris. Pellets from several plates processed in parallel were combined and washed once with RLT buffer and four times with TE buffer (10 mM Tris/HCl pH 8, 1 mM EDTA pH 8) to remove residual contaminations of the host cells. Staphylococcal cell pellets were flashfrozen in liquid nitrogen and stored at −70°C until cell disruption. 58
Maren Depke Material and Methods Pathogen Gene Expression Profiling Bacterial cell disruption For cell disruption the bacterial cell pellet was resuspended on ice in 60 µl TE (10 mM Tris/HCl pH 8, 1 mM EDTA pH 8) supplemented with 20 mM NaN 3 and transferred together with 0.5 ml TRIZOL (Invitrogen, Karlsruhe, Germany) to liquid nitrogen cooled Teflon vessels. Cells were disintegrated mechanically at 2600 rpm for 2 min in a bead mill (Mikrodismembrator S, B. Braun Biotech International GmbH, Melsungen, Germany; now part of Sartorius AG, Göttingen, Germany). Another 0.5 ml of TRIZOL was added and the frozen lysate was allowed to thaw. In total, the liquid lysate was incubated at room temperature for 10 min and afterwards flashfrozen in liquid nitrogen and stored −70°C until RNA preparation. RNA preparation The lysates from bead mill disruption were thawed at room temperature. Chloroform was added (200 µl chloroform / 1 ml TRIZOL), samples were shaken vigorously for 15 s and incubated at room temperature for 5 min. Organic and aqueous phase were separated by centrifugation (12000 x g, 15 min, 4°C) and RNA was precipitated from the aqueous phase with 500 µl isopropanol (2-propanol) / 1 ml TRIZOL overnight at −20°C. After two washes with −20°C precooled 80 % ethanol the RNA was dried at room temperature and solved in nuclease-free water (Ambion Inc., Austin, TX, USA, now part of Applied Biosystems, Foster City, CA, USA). DNase treatment was only possible for control samples because the RNA yield of internalized staphylococci was near the minimum amount needed for tiling array hybridization. For controls, DNase digestion and RNA purification was performed as described before (see Material and Methods/Growth Media Comparison Study, page 56). The concentration was determined photometrically (NanoDrop ND-1000, NanoDrop Technologies, Wilmington, DE, USA) and the quality was checked with an Agilent 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA, USA). 59
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Maren Depke<br />
Material and Methods<br />
Pathogen Gene Expression Pr<strong>of</strong>iling<br />
Bacterial cell disruption<br />
For cell disruption the bacterial cell pellet was resuspended on ice in 60 µl TE (10 mM Tris/HCl<br />
pH 8, 1 mM EDTA pH 8) supplemented with 20 mM NaN 3 and transferred together with 0.5 ml<br />
TRIZOL (Invitrogen, Karlsruhe, Germany) to liquid nitrogen cooled Teflon vessels. Cells were<br />
disintegrated mechanically at 2600 rpm for 2 min in a bead mill (Mikrodismembrator S, B. Braun<br />
Biotech International GmbH, Melsungen, Germany; now part <strong>of</strong> Sartorius AG, Göttingen,<br />
Germany). Another 0.5 ml <strong>of</strong> TRIZOL was added and the frozen lysate was allowed to thaw. In<br />
total, the liquid lysate was incubated at room temperature for 10 min and afterwards flashfrozen<br />
in liquid nitrogen and stored −70°C until RNA preparation.<br />
RNA preparation<br />
The lysates from bead mill disruption were thawed at room temperature. Chlor<strong>of</strong>orm was<br />
added (200 µl chlor<strong>of</strong>orm / 1 ml TRIZOL), samples were shaken vigorously for 15 s and incubated<br />
at room temperature for 5 min. Organic and aqueous phase were separated <strong>by</strong> centrifugation<br />
(12000 x g, 15 min, 4°C) and RNA was precipitated from the aqueous phase with 500 µl<br />
isopropanol (2-propanol) / 1 ml TRIZOL overnight at −20°C. After two washes with −20°C precooled<br />
80 % ethanol the RNA was dried at room temperature and solved in nuclease-free water<br />
(Ambion Inc., Austin, TX, USA, now part <strong>of</strong> Applied Biosystems, Foster City, CA, USA).<br />
DNase treatment was only possible for control samples because the RNA yield <strong>of</strong> internalized<br />
staphylococci was near the minimum amount needed for tiling array hybridization. For controls,<br />
DNase digestion and RNA purification was performed as described before (see Material and<br />
Methods/Growth Media Comparison Study, page 56). The concentration was determined<br />
photometrically (NanoDrop ND-1000, NanoDrop Technologies, Wilmington, DE, USA) and the<br />
quality was checked with an Agilent 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA,<br />
USA).<br />
59
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