microarray data is deposited at the ncbi gene expression omnibus ( geo ) database under geo series accession number gse52552 ( http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=gse52552 ) .
to gain insight in the mechanisms of complement resistance in m. catarrhalis , the transcriptional response of m. catarrhalis strain bbh18 , a complement resistant isolate , , upon exposure to 10% pooled normal human serum ( nhs ) was analyzed by microarray expression profiling .
this concentration was chosen because test experiments demonstrated that a m. catarrhalis bbh18 gene deletion mutant lacking the key complement resistance factor uspa2h   was rapidly killed in 10% nhs ( data not shown ) . to reduce the effect of day to day variation , two fully independent microarray expression profiling
experiments were performed , indicated hereafter as experiments a and b ( table  1 ) .
m. catarrhalis bbh18 was pre - cultured overnight on brain heart infusion ( bhi ) agar plates at 37  c in an atmosphere containing 5% co2 .
bacteria were harvested from plates and resuspended in pbs supplemented with 0.15% gelatin ( pbs - g ) .
next , 5  ml bhi medium was inoculated to an od600  nm of ~  0.075 and cultured until mid - log phase ( od600  nm of ~  1.0 ) , obtaining cultures with a density of 6    10  cfu / ml . of this culture ,
4  ml was harvested by centrifugation , washed in 16  ml pbs - g , and resuspended in 20  ml pbs - g with 0.2  mm mgcl2 and 1  mm cacl2 ( ca and mg ) .
5.5  ml of this suspension was mixed with 5.5  ml of either pbs - g ( ca and mg ) ( control ; experiment a ; n  =  3 , experiment b ; n  =  2 ) or with 20% pooled nhs ( gti diagnostics ) in pbs - g ( ca and mg ) ( experiment a ; n  =  3 , experiment b ; n  =  3 ) and incubated at 37  c with agitation ( 200  rpm ) .
after 0 , 30 ( experiment a only ) , and 60  min , a 3.5  ml aliquot was taken , harvested by centrifugation , and immediately treated with 1  ml of rnaprotect bacteria reagent ( qiagen ) .
total rna was isolated using the rneasy mini kit ( qiagen ) according to manufacturer 's instructions .
co - purified genomic dna was removed by a double dnase treatment using the dna - free kit ( ambion ) .
five micrograms of total rna obtained from samples that were exposed for 60  min to nhs or control conditions was used for microarray expression profiling . the rna was reverse transcribed and labeled for microarray hybridization according to standard nimblegen gene expression array protocols .
two micrograms of cy3-labeled cdna probes was hybridized overnight at 42  c to custom - designed nimblegen m. catarrhalis bbh18 expression arrays ( 4 plex arrays ; 72  k ) and subsequently washed ; all procedures were performed according to manufacturer 's instructions .
array images were acquired with a nimblegen ms200 scanner , and images were first processed with nimblescan software . raw expression data ( .pair files )
were processed using arraystar ( dnastar ) and normalized using the quantile rma method yielding an average signal intensity per cds . to identify genes
differentially expressed in 10% nhs compared to the control condition , statistical analysis was executed using cybert unpaired two - condition data analysis ( http://cybert.ics.uci.edu/ ) .
genes were considered to be differentially expressed when expression was increased or decreased by >  2.5-fold and at a bonferroni corrected p  <  0.05 . to eliminate genes with no or very low expression from our analysis , we generated frequency plots of log2 average probe signal intensity per gene ; two distributions could be observed ( data not shown ) .
based on these frequency plots we eliminated genes with an average normalized probe signal intensity of log2  <  6 in all tested conditions from further analysis . using these criteria we identified 84 genes with a >  2.5 fold increased expression and 134 genes that showed a >  2.5 fold reduced expression in 10% nhs compared to incubation in pbs - g ( nhs dilution buffer ) . to determine if exposure to 10% nhs resulted in the increased expression of genes grouped in the same functional classes , potential enrichment was analyzed with the fishers exact ( one - tail ) test and corrected for multiple testing according to storey and tibshirani . to validate microarray expression data , real - time quantitative pcr ( rt - qpcr ) analysis was performed for a selection of 18 genes on samples obtained after 60  min exposure to nhs or pbs - g ( experiment b ) .
a sub - selection of 11 genes was tested on samples obtained after 30  min ( experiment a ) ( table  1 ) .
dna - free total rna ( 500  ng ) was reverse transcribed into cdna using the iscript cdna synthesis kit ( bio - rad ) .
rt - qpcr was performed in duplicate for each sample using ssoadvanced sybr green supermix ( bio - rad ) and analyzed on a cfx96 real - time pcr machine ( bio - rad ) .
expression fold - changes ( nhs versus pbs - g ) between the different groupings were calculated using the ct method and the gyra gene was used for normalization .
correlation between microarray and rt - qpcr data was assessed with a two - tailed pearson correlation test using graphpad prism software .
to gain insight in the mechanisms of complement resistance in m. catarrhalis , the transcriptional response of m. catarrhalis strain bbh18 , a complement resistant isolate , , upon exposure to 10% pooled normal human serum ( nhs ) was analyzed by microarray expression profiling .
this concentration was chosen because test experiments demonstrated that a m. catarrhalis bbh18 gene deletion mutant lacking the key complement resistance factor uspa2h   was rapidly killed in 10% nhs ( data not shown ) . to reduce the effect of day to day variation , two fully independent microarray expression profiling
experiments were performed , indicated hereafter as experiments a and b ( table  1 ) .
m. catarrhalis bbh18 was pre - cultured overnight on brain heart infusion ( bhi ) agar plates at 37  c in an atmosphere containing 5% co2 .
bacteria were harvested from plates and resuspended in pbs supplemented with 0.15% gelatin ( pbs - g ) .
next , 5  ml bhi medium was inoculated to an od600  nm of ~  0.075 and cultured until mid - log phase ( od600  nm of ~  1.0 ) , obtaining cultures with a density of 6    10  cfu / ml . of this culture ,
4  ml was harvested by centrifugation , washed in 16  ml pbs - g , and resuspended in 20  ml pbs - g with 0.2  mm mgcl2 and 1  mm cacl2 ( ca and mg ) .
5.5  ml of this suspension was mixed with 5.5  ml of either pbs - g ( ca and mg ) ( control ; experiment a ; n  =  3 , experiment b ; n  =  2 ) or with 20% pooled nhs ( gti diagnostics ) in pbs - g ( ca and mg ) ( experiment a ; n  =  3 , experiment b ; n  =  3 ) and incubated at 37  c with agitation ( 200  rpm ) .
after 0 , 30 ( experiment a only ) , and 60  min , a 3.5  ml aliquot was taken , harvested by centrifugation , and immediately treated with 1  ml of rnaprotect bacteria reagent ( qiagen ) .
total rna was isolated using the rneasy mini kit ( qiagen ) according to manufacturer 's instructions .
co - purified genomic dna was removed by a double dnase treatment using the dna - free kit ( ambion ) .
five micrograms of total rna obtained from samples that were exposed for 60  min to nhs or control conditions was used for microarray expression profiling . the rna was reverse transcribed and labeled for microarray hybridization according to standard nimblegen gene expression array protocols .
two micrograms of cy3-labeled cdna probes was hybridized overnight at 42  c to custom - designed nimblegen m. catarrhalis bbh18 expression arrays ( 4 plex arrays ; 72  k ) and subsequently washed ; all procedures were performed according to manufacturer 's instructions .
array images were acquired with a nimblegen ms200 scanner , and images were first processed with nimblescan software . raw expression data ( .pair files )
were processed using arraystar ( dnastar ) and normalized using the quantile rma method yielding an average signal intensity per cds . to identify genes
differentially expressed in 10% nhs compared to the control condition , statistical analysis was executed using cybert unpaired two - condition data analysis ( http://cybert.ics.uci.edu/ ) .
genes were considered to be differentially expressed when expression was increased or decreased by >  2.5-fold and at a bonferroni corrected p  <  0.05 . to eliminate genes with no or very low expression from our analysis , we generated frequency plots of log2 average probe signal intensity per gene ;
based on these frequency plots we eliminated genes with an average normalized probe signal intensity of log2  <  6 in all tested conditions from further analysis . using these criteria we identified 84 genes with a >  2.5 fold increased expression and 134 genes that showed a >  2.5 fold reduced expression in 10% nhs compared to incubation in pbs - g ( nhs dilution buffer ) . to determine
if exposure to 10% nhs resulted in the increased expression of genes grouped in the same functional classes , potential enrichment was analyzed with the fishers exact ( one - tail ) test and corrected for multiple testing according to storey and tibshirani .
to validate microarray expression data , real - time quantitative pcr ( rt - qpcr ) analysis was performed for a selection of 18 genes on samples obtained after 60  min exposure to nhs or pbs - g ( experiment b ) .
a sub - selection of 11 genes was tested on samples obtained after 30  min ( experiment a ) ( table  1 ) .
dna - free total rna ( 500  ng ) was reverse transcribed into cdna using the iscript cdna synthesis kit ( bio - rad ) .
rt - qpcr was performed in duplicate for each sample using ssoadvanced sybr green supermix ( bio - rad ) and analyzed on a cfx96 real - time pcr machine ( bio - rad ) .
expression fold - changes ( nhs versus pbs - g ) between the different groupings were calculated using the ct method and the gyra gene was used for normalization .
correlation between microarray and rt - qpcr data was assessed with a two - tailed pearson correlation test using graphpad prism software .
 the complement system is an important part of the innate defense against invading pathogens ( blom et al . , 2009 ; [ 1 ] ) . 
 the ability to resist complement - mediated killing is considered to be an important virulence trait for the human - restricted respiratory tract pathogen moraxella catarrhalis , as most disease - associated m. catarrhalis isolates are complement - resistant ( wirth et al . 
 , 2007 ; [ 2 ] ) . 
 here we provide a detailed overview of the experimental methods that we have used to study the molecular basis of m. catarrhalis complement - resistance by transcriptome profiling of the bacterium upon exposure to 10% normal human serum ( nhs ) , associated with the study of de vries et al . published in molecular microbiology in 2014 [ 3 ] . 
Direct link to deposited data
Experimental design, materials and methods
Exposure of 
Microarray gene expression analysis
Validation of microarray data by RT-qPCR analysis