genomewide characterization of host-pathogen interactions by ...

More documents

Recommendations

Info

Maren Depke Material and Methods Pathogen Gene Expression Profiling Tiling Array Expression Profiling Tiling array design The “080604_SA_JH_Tiling” array was designed by Hanne Jarmer (Center for Biological Sequence Analysis, Department of Systems Biology, Technical University of Denmark, Lyngby, Denmark) using algorithms, which have recently been published in the context of a B. subtilis tiling array (Rasmussen et al. 2009). In brief, iso-thermal probes of 45 nucleotides (nt) to 65 nt were designed to cover the whole genome of S. aureus. Probes were arranged in 22 nt intervals on each strand and possessed an offset of 11 nt between the strands. The array design for S. aureus was part of the international cooperation EU-IP-FP6-project BaSysBio (LSHG-CT2006- 037469) which is funded by the European Commission and coordinated by Philippe Noirot (INRA, Mathématique Informatique et Génome, Jouy-en-Josas, France). The sequences were synthesized on quartz wafers by NimbleGen (Roche NimbleGen, Madison, WI, USA) in a custom, high-density DNA array format by applying the Maskless Array Synthesizer (MAS) technology in combination with photo-mediated synthesis chemistry. The basic principle of the synthesis is a solid-state array of miniature aluminum mirrors which direct UV-light at the place where the next reaction steps should occur. An UV-labile protection group is separated from the nascent oligonucleotide and releases a reactive site for binding of the next nucleotide. Thus, the synthesis of the oligonucleotide sequences requires m x 4 reaction steps, where m is the length of the oligonucleotide (number of nucleotides) and at each intermediate length step the four possible nucleotides A, T, C, and G will be added in a single, separate reaction. Tiling array hybridization The tiling array hybridization was performed at NimbleGen (Roche NimbleGen, Madison, WI, USA) according to standard protocols. Briefly, 10 µg of high quality RNA samples were reverse transcribed to cDNA in the presence of actinomycin D with subsequent alkaline RNA hydrolysis. Precipitated and resolved cDNA was labeled with Cy3 dye via NHS-Ester Dye Coupling Reaction. After spin-column based purification and quality control, labeled cDNA was hybridized together with appropriate controls to tiling arrays of the 080604_SA_JH_Tiling design. Arrays were washed and scanned and raw intensity data of tiling probes were provided to the customer. Tiling array raw data analysis The raw data analysis and condensing of probe data to transcripts/genes was performed by Pierre Nicolas, Aurélie Leduc, and Philippe Bessières (INRA, Mathématique Informatique et Génome, Jouy-en-Josas, France). Intensity values for annotated genes were derived from the individual probe data by calculating the median of the probes located within the genomic coordinates of these genes. Analysis of hybridization signal, identification of transcription start and end boundaries and by this means segmentation into transcriptional units was executed by a novel algorithm based on a hidden Markov model, which allows to identify new, formerly unknown or non-annotated transcripts (Nicolas et al. 2009). 60
Maren Depke Material and Methods Pathogen Gene Expression Profiling Tiling array expression data analysis Condensed, linear space gene expression values were imported via the Gene Expression Text Loader (GETL) into the Rosetta Resolver software (Rosetta Biosoftware, Seattle, WA, USA) for data analysis. Inter-chip median-scaling (normalization) and detrending (Rosetta Resolver proprietary algorithm) were applied in the experiment definitions (ED) to allow direct comparison of values from different arrays. Groups were compared in log-transformed space using textbook one-way ANOVA with Benjamini-Hochberg False Discovery Rate multiple testing correction and p* < 0.05 was regarded as significant. An absolute fold change cutoff of 2 was applied. 61
Page 1 and 2:
G E N O M E W I D E C H A R A C T E
Page 3 and 4:
Maren Depke C O N T E N T S GENOMEW
Page 5 and 6:
Maren Depke Z U S A M M E N F A S S
Page 7 and 8:
Maren Depke Zusammenfassung der Dis
Page 9 and 10: Maren Depke S U M M A R Y O F D I S
Page 11 and 12: Maren Depke Summary of Dissertation
Page 13 and 14: Maren Depke I N T R O D U C T I O N
Page 15 and 16: Maren Depke Introduction Besides op
Page 17 and 18: Maren Depke Introduction Extracellu
Page 19 and 20: Maren Depke Introduction with heigh
Page 21 and 22: Maren Depke Introduction 2005). SaP
Page 23 and 24: Maren Depke Introduction contains a
Page 25 and 26: Maren Depke Introduction via fibron
Page 27 and 28: Maren Depke Introduction cathelicid
Page 29 and 30: Maren Depke Introduction shock are
Page 31 and 32: Maren Depke Introduction STUDIES OF
Page 33 and 34: Maren Depke Introduction are effect
Page 35 and 36: Maren Depke Introduction cell stimu
Page 37 and 38: Maren Depke Introduction channel CF
Page 39 and 40: Maren Depke M A T E R I A L A N D M
Page 41: Maren Depke Material and Methods Li
Page 44 and 45: Maren Depke Material and Methods Ki
Page 47 and 48: Maren Depke Material and Methods GE
Page 49: Maren Depke Material and Methods Ge
Page 52 and 53: Maren Depke Material and Methods Ho
Page 54 and 55: Maren Depke Material and Methods Ho
Page 56 and 57: Maren Depke Material and Methods Pa
Page 58 and 59: Maren Depke Material and Methods Pa
Page 63 and 64: corticosterone [pg/ml plasma] corti
Page 65 and 66: Maren Depke Results Liver Gene Expr
Page 67 and 68: lood glucose levels [nmol/l] resist
Page 69 and 70: LBP [ng/ml] CPR [ng/ml] Maren Depke
Page 71 and 72: Maren Depke Results Liver Gene Expr
Page 73 and 74: liver weight [g] Maren Depke Result
Page 75 and 76: infection rate cfu / 10 mg tissue c
Page 77 and 78: Maren Depke Results Kidney Gene Exp
Page 79 and 80: Table R.2.1: Genes displaying stati
Page 81 and 82: Maren Depke BR1 sign DsigB vs wt BR
Page 91 and 92: Maren Depke Results GENE EXPRESSION
Page 93 and 94: Maren Depke Results Gene Expression
Page 95 and 96: log2(ratio C57BL/6 / BALB/c) at IFN
Page 109: Maren Depke Results Gene Expression
Page 112 and 113:
Maren Depke Results Host Cell Gene
Page 114 and 115:
Page 116 and 117:
Page 118 and 119:
Page 120 and 121:
Page 122 and 123:
Page 124 and 125:
Page 126 and 127:
Page 128 and 129:
Page 130 and 131:
Page 132 and 133:
Maren Depke Results Pathogen Gene E
Page 134 and 135:
Page 136 and 137:
Page 138 and 139:
S. aureus RN1HG sample conditions a
Page 140 and 141:
Page 142 and 143:
Page 144 and 145:
Page 146 and 147:
Page 148 and 149:
mean normalized intensity mean norm
Page 150 and 151:
Page 152 and 153:
Page 154 and 155:
Page 156 and 157:
Page 158 and 159:
mean normalized intensity Maren Dep
Page 160 and 161:
Page 162 and 163:
Page 164 and 165:
Page 166 and 167:
Page 168 and 169:
log 2 (ratio) log 2 (ratio) log 2 (
Page 171 and 172:
Maren Depke D I S C U S S I O N A N
Page 173 and 174:
Maren Depke Discussion and Conclusi
Page 175 and 176:
Page 177 and 178:
Page 179 and 180:
Page 181 and 182:
Page 183 and 184:
Page 185 and 186:
Page 187 and 188:
Page 189 and 190:
Page 191 and 192:
Page 193 and 194:
Page 195 and 196:
Page 197 and 198:
Page 199 and 200:
Page 201 and 202:
Page 203 and 204:
Page 205:
Page 208 and 209:
Maren Depke References Athanasopoul
Page 210 and 211:
Maren Depke References Byrne GI, Le
Page 212 and 213:
Maren Depke References Demling R. T
Page 214 and 215:
Maren Depke References Foss GS, Pry
Page 216 and 217:
Maren Depke References Hagopian K,
Page 218 and 219:
Maren Depke References Jin T, Bokar
Page 220 and 221:
Maren Depke References Kwan T, Liu
Page 222 and 223:
Maren Depke References Luster AD, U
Page 224 and 225:
Maren Depke References Namiki S, Na
Page 226 and 227:
Maren Depke References Puccetti P.
Page 228 and 229:
Maren Depke References Siewert E, B
Page 230 and 231:
Maren Depke References van den Akke
Page 232 and 233:
Maren Depke References Yang XL, Sch
Page 235:
Maren Depke A F F I D A V I T / E R
Page 238 and 239:
Maren Depke Acknowledgments Kidney
show all

genomewide characterization of host-pathogen interactions by ...

You also want an ePaper? Increase the reach of your titles

Delete template?

Save as template?