illumina hi - seq 2500 data is deposited at the ncbi gene expression omnibus ( geo ) database under geo series and is available at the following link : http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=gse64507 . s2 cells were maintained at 28  c in schneider 's drosophila medium ( invitrogen ) , supplemented with 10% heat - inactivated fetal bovine serum ( life technologies ) . for bacterial stimulation , heat - killed s. typhimurium was added to s2 cells at a final concentration of 10  cells / ml . at 0 , 30  min , 1  h , and 4  h after stimulation , 3    10  s2 cells were harvested each time point , and cross - linked by incubating with 1% formaldehyde at room temperature for 10  min . to stop the protein - dna cross - links , 125  mm glycine was added . nuclei were isolated by re - suspending cells in nuclei isolation buffer ( 0.3  m sucrose , 2  mm mgoac2 , 1  mm cacl2 , 1% nonidet p-40 , 10  mm hepes , ph  7.6 ) and spinning at 800  g for 10  min . s2 nuclei were digested with 40  u / ml micrococcal nuclease ( mnase ) ( worthington biochemical corp ) in mnase cleavage buffer ( 4  mm cacl2 , 25  mm kcl , 4  mm mgcl2 , 12.5% glycerol , 50  mm hepes , ph  7.4 ) at 37  c for 5  min . rna was removed by treating samples with 100  g / ml rnase a ( thermo scientific ) at 55  c for 10  min . samples were incubated at 65  c over night with 0.2  g/l proteinase k ( worthington biochemical corp ) and 1% sodium dodecyl sulfate to remove the cross - links . dna was purified by phenol  chloroform extraction and ethanol precipitation . to isolate mononucleosomal dna , the area of the gel containing mononucleosomal dna ( 200  bps and below ) was excised and purified by the freeze - squeeze method , , . libraries of mononucleosomal dna were prepared using nebnext ultra dna library prep kit for illumina ( neb # e7370s / l ) , starting with 30  ng of mononucleosomal dna for each reaction . the samples were end - prepared , adaptor - ligated and indexed ( neb # e7335s / l , neb # e7500s / l ) according to the manufacturer 's instructions , then purified with ampure xp beads ( beckman coulter , inc . # a63881 ) without size selection . the quantity and quality of the library were checked with qubit 2.0 fluorometer ( life sciences ) and agilent 2100 bioanalyzer ( agilent technologies ) resulting libraries were plated using the illumina cbot and run on the illumina hiseq 2500 platform configured for 50  bp paired - end reads . nucleosome occupancy was calculated by reads per million that mapped to the fly genome with bedtools 2.17 . analyses of nucleosome occupancy were performed in r environment using our lab - developed software package : rage.r . we created heatmaps for each time point by plotting the nucleosome occupancy of all fly genes and centering the genes at the tss . we found a significant loss of nucleosome occupancy at the 4  h time point post s. typhimurium stimulation ( k - s test 2.2    10 ) ( fig .  1 ) . these results suggest that widespread nucleosome loss may be an instrumental part of genome regulation during the immune response to s. typhimurium . s2 cells were maintained at 28  c in schneider 's drosophila medium ( invitrogen ) , supplemented with 10% heat - inactivated fetal bovine serum ( life technologies ) . for bacterial stimulation , heat - killed s. typhimurium was added to s2 cells at a final concentration of 10  cells / ml . at 0 , 30  min , 1  h , and 4  h after stimulation , 3    10  s2 cells were harvested each time point , and cross - linked by incubating with 1% formaldehyde at room temperature for 10  min . to stop the protein - dna cross - links , 125  mm glycine was added . nuclei were isolated by re - suspending cells in nuclei isolation buffer ( 0.3  m sucrose , 2  mm mgoac2 , 1  mm cacl2 , 1% nonidet p-40 , 10  mm hepes , ph  7.6 ) and spinning at 800  g for 10  min . s2 nuclei were digested with 40  u / ml micrococcal nuclease ( mnase ) ( worthington biochemical corp ) in mnase cleavage buffer ( 4  mm cacl2 , 25  mm kcl , 4  mm mgcl2 , 12.5% glycerol , 50  mm hepes , ph  7.4 ) at 37  c for 5  min . rna was removed by treating samples with 100  g / ml rnase a ( thermo scientific ) at 55  c for 10  min . samples were incubated at 65  c over night with 0.2  g/l proteinase k ( worthington biochemical corp ) and 1% sodium dodecyl sulfate to remove the cross - links . dna was purified by phenol  chloroform extraction and ethanol precipitation . to isolate mononucleosomal dna , the area of the gel containing mononucleosomal dna ( 200  bps and below ) was excised and purified by the freeze - squeeze method , , . libraries of mononucleosomal dna were prepared using nebnext ultra dna library prep kit for illumina ( neb # e7370s / l ) , starting with 30  ng of mononucleosomal dna for each reaction . e7500s / l ) according to the manufacturer 's instructions , then purified with ampure xp beads ( beckman coulter , inc . # a63881 ) without size selection . the quantity and quality of the library were checked with qubit 2.0 fluorometer ( life sciences ) and agilent 2100 bioanalyzer ( agilent technologies ) resulting libraries were plated using the illumina cbot and run on the illumina hiseq 2500 platform configured for 50  bp paired - end reads . nucleosome occupancy was calculated by reads per million that mapped to the fly genome with bedtools 2.17 . analyses of nucleosome occupancy were performed in r environment using our lab - developed software package : rage.r . we created heatmaps for each time point by plotting the nucleosome occupancy of all fly genes and centering the genes at the tss . we found a significant loss of nucleosome occupancy at the 4  h time point post s. typhimurium stimulation ( k - s test 2.2    10 ) ( fig .  1 ) . these results suggest that widespread nucleosome loss may be an instrumental part of genome regulation during the immune response to s. typhimurium .