genomewide characterization of host-pathogen interactions by ...

Maren Depke
Material and Methods
KIDNEY GENE EXPRESSION PATTERN IN AN
IN VIVO INFECTION MODEL
In vivo infection model, organ harvesting, and group size [performed by Tina Schäfer]
Female BALB/c mice (Charles River, Sulzfeld, Germany) were infected i. v. with 5.0E+07 colony
forming units (cfu; first biological replicate, BR1) and 7.0E+07 cfu (second biological replicate,
BR2) S. aureus RN1HG or 5.0E+07 cfu (first biological replicate) and 8.0E+07 cfu (second biological
replicate) of its isogenic sigB mutant S. aureus RN1HG ΔsigB. The third group in this study
comprised sham-infected mice which received an injection of 100 µl physiological saline solution
(performed only in the second biological replicate of the experiment). After 4 days (first biological
replicate) or 5 days (second biological replicate) mice were sacrificed and kidneys were explanted
immediately afterwards, flash-frozen in liquid nitrogen and stored at −80°C.
Each of the three experimental groups was comprised of 5 independent samples per biological
replicate (originating from kidneys of 5 mice) except for the group of infection with S. aureus
RN1HG in the first biological replicate, which only consisted of 4 samples. In total, 24 samples
were analyzed in this study.
Tissue disruption
In constant submersion in liquid nitrogen in a mortar, both kidneys of the mice were ground
into very small pieces, but not into powder. By this means it was possible to yield a homogenous
tissue mix which allowed accurate estimation of infection rate. As the tissue was not completely
disrupted into powder, it was still possible to handle the frozen tissue mix e. g. during aliquoting
without the risk of sample thawing.
DNA preparation
Small aliquots of disrupted tissue were added to Lysing Matrix D tubes (MP Biomedicals,
Solon, OH, USA) which contain 1.4 mm diameter ceramic spheres. Tissue was completely
disrupted in 190 µl of 42 mM EDTA using a FastPrep FP120 (Thermo Fisher Scientific Inc.,
Waltham, MA, USA) at level 6.5 for 20 s. After short cooling on ice, samples were digested with
250 µg/µl lysostaphin (AMBI PRODUCTS LLC, Lawrence, NY, USA) for 45 min at 37°C to ensure
complete lysis of staphylococci in the infected tissue. The following DNA preparation employed
the Wizard Genomic DNA Purification Kit (Promega Corp., Madison, WI, USA) with minor
modifications to the manufacturer’s protocol. The tissue lysate (200 µl) was mixed with 830 µl of
Nuclei Lysis Solution (Promega) and incubated for 5 min at 80°C. Subsequently, samples were
cooled for 5 min on ice and digested with 19.4 ng/µl RNase A (Promega) for 30 min at 37°C.
Samples were again cooled on ice for 5 min and afterwards processed at room temperature.
Lysates were transferred to new 1.5-ml-tubes, 345 µl of Protein Precipitation Solution were
added, samples were vortexed for 20 s and protein was precipitated for 10 min on ice. The
solution was cleared by two centrifugation steps at 20000 x g for 4 min (room temperature). The
final clear supernatant was divided into two aliquots of 620 µl each and the DNA was precipitated
by addition of 470 µl of isopropanol (2-propanol) and mixing by gentle inversion. After
centrifugation for 2 min at 16000 x g (room temperature) the DNA pellet was washed with 600 µl
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