overnight cultures of s. aureus newman and its ccpe deletion mutant ( ccpe ) were washed and diluted 100-fold in fresh tsb medium ( without glucose ) in a 20-ml tube with a tube volume - to - medium volume ratio of 5:1 and in triplicates .
the liquid culture was grown at 37  c for about 6  h ( od600    5.0 ) with shaking , 250  rpm of aeration .
total rna was immediately stabilized with an rnaprotect bacteria reagent ( qiagen , valencia , ca ) following the manufacturer 's instructions .
the s. aureus cells were harvested by centrifugation and suspended in 1  ml 1   te ( 10  mm tris , ph  8.5 , 1  mm edta ) buffer containing lysostaphin ( final concentration of 50  g / ml ) , suspensions were incubated at 37  c for 30  min .
the bacteria were subsequently disrupted by mechanical disruption ( fastprep-24 instrument from mp biomedicals ) and rna extraction was performed as described before ,   through the use of an rneasy mini kit ( qiagen , valencia , ca ) following the manufacturer 's instructions .
the concentration of rna was checked on a spectrophotometer and the quality of rna was checked using formaldehyde agarose gel electrophoresis and agilent rna analysis kit ( agilent technologies ) .
the cdna preparation and labeling were performed by capitalbio corp ( http://www.capitalbio.com/index.asp , beijing , china ) according to the manufacturer ( affymetrix , santa clara , ca ) with some minor modifications . in short , total rna ( 10  g )
was mixed with 10  l random primers ( 75  ng/l ) and 2  l diluted poly - a rna controls and nuclease free water was added if necessary to keep the final volume of the annealing mixture to 30  l according to the standard affymetrix protocol ( genechip expression analysis technical manual , chapter 4 prokaryotic target preparation . 702232 , rev.3 ) .
the annealing mixture was kept at 70  c for 10  min and then at 25  c for 10  min , and hold at 4  c .
30  l of master mix for cdna synthesis [ consisting of 12  l 5   first strand buffer , 6  l 100  mm dtt , 3  l 10  mm dntps , 1.5  l superase - in ( 20  u/l ) rnase inhibitor and 7.5  l superscript ii reverse transcriptase ( 200  u/l ) ] was added to the annealing mixture and the reaction mix was incubated at the following temperatures : 25  c for 10  min ; 37  c for 60  min ; 42  c for 60  min ; 70  c for 10  min ( in order to inactive superscript ii ) and chill to 4  c . following the incubation ,
the mrna from the reaction mixture was degraded by adding 20  l of 1  n naoh and placing the reactions at 65  c for 30  min . to neutralize the naoh in reactions , 20  l of 1  n hcl was added .
the cdna mixture was purified by using a minelute pcr purification kit ( qiagen ) and the purified cdna product was analyzed by reading the absorbance under 260  nm for the quantification ( 1.0  a260  unit  =  33  g / ml of single - stranded dna ) .
cdna was end - labeled ( 3 ) using a genechip labeling reagent ( affymetrix , p / n 900542 ) and following the manufacturer 's protocol .
the labeled cdna ( 0.57.0  g ) was then hybridized to the affymetrix genechip s. aureus genome array ( affymetrix , cat .
900514 ) for 16  h at 50  c through the use of the genechip hybridization oven at 60  rpm . washing , staining , and scanning were performed using the affymetrix genechip system at capitalbio corp ( http://www.capitalbio.com/index.asp , beijing , china ) .
microarray slides were scanned and pre - analyzed using genechip operating software ( gcos ) version 1.2 ( affymetrix ) and genespring gx version 7.3 ( agilent technologies , inc .
the microarray data were further normalized using robust multi - array average ( rma ) , .
gene expression analysis was performed using three biological replicate mrna samples for each strain which were analyzed with sam ( significance analysis of microarrays ) software .
for differentially expressed genes , fold change   2 or   0.5 and q - value   5% was used as standard .
overnight cultures of s. aureus newman and its ccpe deletion mutant ( ccpe ) were washed and diluted 100-fold in fresh tsb medium ( without glucose ) in a 20-ml tube with a tube volume - to - medium volume ratio of 5:1 and in triplicates .
the liquid culture was grown at 37  c for about 6  h ( od600    5.0 ) with shaking , 250  rpm of aeration .
total rna was immediately stabilized with an rnaprotect bacteria reagent ( qiagen , valencia , ca ) following the manufacturer 's instructions .
the s. aureus cells were harvested by centrifugation and suspended in 1  ml 1   te ( 10  mm tris , ph  8.5 , 1  mm edta ) buffer containing lysostaphin ( final concentration of 50  g / ml ) , suspensions were incubated at 37  c for 30  min .
the bacteria were subsequently disrupted by mechanical disruption ( fastprep-24 instrument from mp biomedicals ) and rna extraction was performed as described before ,   through the use of an rneasy mini kit ( qiagen , valencia , ca ) following the manufacturer 's instructions .
the concentration of rna was checked on a spectrophotometer and the quality of rna was checked using formaldehyde agarose gel electrophoresis and agilent rna analysis kit ( agilent technologies ) .
the cdna preparation and labeling were performed by capitalbio corp ( http://www.capitalbio.com/index.asp , beijing , china ) according to the manufacturer ( affymetrix , santa clara , ca ) with some minor modifications . in short , total rna ( 10  g )
was mixed with 10  l random primers ( 75  ng/l ) and 2  l diluted poly - a rna controls and nuclease free water was added if necessary to keep the final volume of the annealing mixture to 30  l according to the standard affymetrix protocol ( genechip expression analysis technical manual , chapter 4 prokaryotic target preparation .
the annealing mixture was kept at 70  c for 10  min and then at 25  c for 10  min , and hold at 4  c .
30  l of master mix for cdna synthesis [ consisting of 12  l 5   first strand buffer , 6  l 100  mm dtt , 3  l 10  mm dntps , 1.5  l superase - in ( 20  u/l ) rnase inhibitor and 7.5  l superscript ii reverse transcriptase ( 200  u/l ) ] was added to the annealing mixture and the reaction mix was incubated at the following temperatures : 25  c for 10  min ; 37  c for 60  min ; 42  c for 60  min ; 70  c for 10  min ( in order to inactive superscript ii ) and chill to 4  c . following the incubation ,
the mrna from the reaction mixture was degraded by adding 20  l of 1  n naoh and placing the reactions at 65  c for 30  min . to neutralize the naoh in reactions , 20  l of 1  n hcl was added .
the cdna mixture was purified by using a minelute pcr purification kit ( qiagen ) and the purified cdna product was analyzed by reading the absorbance under 260  nm for the quantification ( 1.0  a260  unit  =  33  g / ml of single - stranded dna ) .
cdna was end - labeled ( 3 ) using a genechip labeling reagent ( affymetrix , p / n 900542 ) and following the manufacturer 's protocol .
the labeled cdna ( 0.57.0  g ) was then hybridized to the affymetrix genechip s. aureus genome array ( affymetrix , cat .
900514 ) for 16  h at 50  c through the use of the genechip hybridization oven at 60  rpm . washing , staining , and scanning were performed using the affymetrix genechip system at capitalbio corp ( http://www.capitalbio.com/index.asp , beijing , china ) .
microarray slides were scanned and pre - analyzed using genechip operating software ( gcos ) version 1.2 ( affymetrix ) and genespring gx version 7.3 ( agilent technologies , inc . ,
the microarray data were further normalized using robust multi - array average ( rma ) , .
gene expression analysis was performed using three biological replicate mrna samples for each strain which were analyzed with sam ( significance analysis of microarrays ) software . for differentially expressed genes , fold change   2 or   0.5 and q - value   5% was used as standard .
 the transcriptional regulator ccpe is an important citrate - sensing regulator that modulates metabolic state , virulence factor expression , and bacterial virulence of staphylococcus aureus ( ding et al . , 2014 [ 1 ] ) . 
 in this article , we report detailed methods for genome - wide transcriptional profiling of ccpe - regulated genes generated for the research article  metabolic sensor governing bacterial virulence in staphylococcus aureus  
 ( ding et al . , 2014 [ 1 ] ) . 
 all transcriptional profiling data was deposited to gene expression omnibus ( geo ) database under accession number gse57260 . 
Direct link to deposited data
Experimental design, materials and methods
Bacterial strains and growth conditions
RNA extraction
cDNA preparation and labeling
Hybridization and washing
Microarray data analysis
Conflict of interest