genomewide characterization of host-pathogen interactions by ...

Maren Depke
Material and Methods
Host Cell Gene Expression Pattern in an in vitro Infection Model
515 nm to 545 nm (FITC filter block). All FSC and SSC data were recorded at linear scale, the
fluorescence data were recorded at logarithmic scale with the FACS Diva v5.03 software (Becton
Dickinson). Prior to measurement of bacteria containing eukaryotic S9 cells, the proper function
of the instrument was determined by using Spherotech’s Rainbow calibration particles
(Spherotech, Lake Forest, IL, USA). Prior to sorting, the drop delay was adjusted to >99 % sorting
with the ACCUDROP beads (Becton Dickinson). Sorting was performed from the gate set in FSC
vs. FITC dot plot after eliminating doublets and cell clumps by gating SSC−W vs. SSC−A and FSC−W
vs. FSC−A at a threshold rate up to 3000 cells/s with sort mode purity, which results in a sorted
sample that is highly pure, at the expense of recovery and yield.
For each transcriptome sample, 5.0E+05 to 7.0E+05 cells were collected, whereas for protein
samples approximately 4.0E+06 cells were sorted. During the whole period of sorting and sample
collection, the sample reservoir and the collection tubes were cooled to 4°C. Control samples
were stored on ice during sorting of infected samples. Sorted and control cell suspensions were
centrifuged for 5 min at 500 x g and 4°C. The supernatants were removed and each cell pellet
devoted to transcriptome analysis was lysed in 1 ml TRIZOL (Invitrogen, Karlsruhe, Germany) by
repeated pipetting. After incubation for 10 min at room temperature, the lysate was flash-frozen
in liquid nitrogen and stored −70°C until RNA preparation. The remaining cells after sorting of
samples for transcriptome and proteome analysis were used to acquire information on cell
vitality by propidium iodide staining.
RNA preparation
The lysates 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 and 1 µl 5 mg/ml linear acrylamide (Ambion Inc., Austin, TX, USA, now
part of Applied Biosystems, Foster City, CA, USA) as precipitation aid overnight at −20°C. After
two washes with −20°C pre-cooled 80 % ethanol, the RNA was dried at room temperature and
solved in nuclease-free water (Ambion). 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).
DNA array analysis
The RNA expression profile of host cells was analyzed on GeneChip Human Gene 1.0 ST arrays
(Affymetrix, Santa Clara, CA, USA) using the Whole Transcript (WT) Sense Target Labeling and
Control reagents according to the manufacturer’s instructions. Arrays were washed and stained
in a GeneChip FluidicsStation 450 and scanned with a GeneChip Scanner 3000 (all: Affymetrix).
DNA array data analysis
The array image files (CEL) were first quality controlled in the Expression Console software
(Affymetrix) and then imported into the Rosetta Resolver software (Rosetta Biosoftware, Seattle,
WA, USA) for data analysis. Signals were generated and normalized using the RMA algorithm.
Groups were compared in log-transformed space using error-weighted one-way ANOVA with
Benjamini-Hochberg False Discovery Rate multiple testing correction and p* < 0.01 was regarded
as significant. The control sequences (e. g. negative, positive, polyA, and hybridization controls)
and sequences that were not expressed on all arrays in the selected group comparison (with
p > 0.01 on intensity profile level in Rosetta Resolver software) were not included in statistical
testing.
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