rkshv.219 virus is latently infected in islk.rkshv219 cell line , derived from islk cell line , as previously described by .
rkshv.219 cells were seeded at 5    10 cells into 150-mm dish ( one day prior to induction ) . for induction of kshv reactivation ,
the previous medium is replaced with 1% fbs dmem media containing a final concentration of 0.2  g / ml doxycycline .
kshv reactivation was induced at 0 , 6 , 12 , 24 , and 48  h. the 0  h sample was treated identically to other timepoints , however doxycycline was not added .
two independent biological replicates for each timepoint in islk.rkshv219 cell lines were processed and behaved similar for the nucleosome distribution assay .
following doxycycline induction of kshv reactivation , islk.rkshv219 cells were harvested at 0 , 6 , 12 , 24 , and 48  h at 2.5    10 cells .
media was aspirated and then 1% formaldehyde in pbs was incubated with the cells for 10  min at room temperature , to cross - link the chromatin structure .
125  mm glycine was then added to stop the formaldehyde cross - linking reaction .
nucleus isolation buffer , 0.3  m sucrose , 2  mm mgoac2 , 1  mm cacl2 , 1% nonidet p-40 , 10  mm hepes , and ph  7.8 were then added to islk.rkshv219 cross - linked cells .
the cells were scraped and placed within a 50  ml conical tube , and the nuclei were isolated by centrifugation at 1000  g for 5  min at 4  c .
micrococcal nuclease , mnase , is a internucleosomal cleavage reagent , which does not cleave the dna protected by a nucleosome .
islk.rkshv219 nuclei were digested with a titration of mnase : 4  u / ml , 2  u / ml , 1  u / ml , and 0.5  u / ml ( worthington biochemical corp . ) in mnase cleavage buffer ( 5  mm kcl , 4  mm mgcl2 , 1  mm cacl2 , 12.5% glycerol , and 50  mm tris  cl ( ph  7.4 ) ) .
a titration of mnase concentrations was utilized to ensure nucleosomes that are more and less readily cleaved are equally represented .
the nuclei , following mnase digestion , were treated with 0.2  g/l proteinase k and 1% sodium dodecyl sulfate .
mnase digested dna for all samples was resolved on a 2% agarose gel , and mononucleosomally protected dna , ~  150  bp , was isolated by gel excision .
following gel excision , the mononucleosomally protected dna was purified by electroelution , and for each sample 's mnase titration the mononucleosomally protected dna was combined .
the mononucleosomally protected dna was further purified with phenol  chloroform extraction and precipitated with alcohol .
islk.rkshv219 cells were resuspended in 0.2-g/l proteinase k and 1% sds solution and incubated at 55  c overnight for bare genomic dna isolation .
we utilized a high - resolution dna microarray , which we customized to contain 1  kb upstream and downstream of the transcription start sites of 462 immunity - related genes .
nimblegen 's 12-plex and hd2 design were utilized for the custom dna microarray 100319_hg19_immunity_jd_cgh array .
sequences were repeat - masked around the tsss , and only probes containing unique sequences for both the forward and reverse dna strands were printed on the microarray .
oligonucleotide probes , sized at 60  bp , were tiled on each subarray with a mean spacing of 47  bp overlap .
approximately 180 probes of spacing of 12.5  bp were printed on the microarray for each locus .
the samples were fluorescently labeled : nucleosomally protected dna with cy3 and bare genomic dna ( reference ) with cy5 .
samples were hybridized to the dna microarray according to nimblegen 's instructions ( http://www.nimblegen.com/technology/index.html ) .
the resulting .tiff files were used for microarray data feature extraction , primary data analysis and generation of initial data files using the nimblegen deva software version 1.2 cgh workflow .
specifically , loess spatial correction was applied to the raw data ; background correction was used to determine and adjust the signal data for noise using specifically designed background control probes ; and qspline normalization was applied to compensate for inherent differences in cy3 and cy5 signal .
the ratio between nucleosomal ( cy3 ) and bare genomic cy5 signals was then calculated and stored as a general feature format , .gff
subsequent analysis was performed using a software developed in our laboratory dennislab.r , which runs in the r environment for statistical computing .
dennislab.r was used to generate summary statistics on the range of nucleosome distribution values for each individual chromosome .
finally , we analyzed the nucleosome distribution changes at the 24  hour timepoint on a per chromosome basis , by calculating the t - statistic and p - value ( assuming unequal variance ) for the pairwise comparison of the range of values between all chromosomes . in our previous work
we demonstrated that nucleosome redistributions are widespread , transient , and dna - directed , .
we next wanted to determine if nucleosome redistributions showed chromosome specific changes in a time dependent manner .
we first calculated the range of nucleosome occupancy values for all loci studied on a per chromosome basis for each post - kshv reactivation timepoint ( fig . 
these results comport with our previous observation that the greatest positioning occurs at the 24  hour timepoint , however do not indicate chromosome specificity with respect to increased positioning .
we next wanted to investigate if the nucleosome redistribution changes at the 24  hour timepoint reflected increased or decreased nucleosomal occupancy .
we calculated the median nucleosome occupancy values on each chromosome for all of the loci measured .
we discovered that nucleosome occupancy values change in a chromosome specific manner ( fig . 
chromosomes 18 , 19 , and 22 show the greatest median nucleosome occupancy values at the 24  hour timepoint .
conversely , chromosomes 1 , 2 , 9 , 10 , 12 , 15 , and 17 show the lower median nucleosome occupancy values at the 24  hour timepoint .
we were next interested in calculating the statistical significance of the chromosome specific nucleosome occupancy changes that we had identified at the 24  hour timepoint .
most of the ranges of nucleosome occupancy values are not statistically significantly different from one another ( fig .  1c , yellow highlights ) .
these two chromosomes show clear increases in the nucleosome occupancy relative to the other chromosomes . at the other end of the scale , chromosomes
these results demonstrate that while nucleosomal positioning remains relatively constant between chromosomes at each timepoint , nucleosomal occupancy changes in a chromosome specific manner .
rkshv.219 virus is latently infected in islk.rkshv219 cell line , derived from islk cell line , as previously described by .
rkshv.219 cells were seeded at 5    10 cells into 150-mm dish ( one day prior to induction ) . for induction of kshv reactivation ,
the previous medium is replaced with 1% fbs dmem media containing a final concentration of 0.2  g / ml doxycycline .
kshv reactivation was induced at 0 , 6 , 12 , 24 , and 48  h. the 0  h sample was treated identically to other timepoints , however doxycycline was not added .
two independent biological replicates for each timepoint in islk.rkshv219 cell lines were processed and behaved similar for the nucleosome distribution assay .
following doxycycline induction of kshv reactivation , islk.rkshv219 cells were harvested at 0 , 6 , 12 , 24 , and 48  h at 2.5    10 cells .
media was aspirated and then 1% formaldehyde in pbs was incubated with the cells for 10  min at room temperature , to cross - link the chromatin structure .
125  mm glycine was then added to stop the formaldehyde cross - linking reaction .
nucleus isolation buffer , 0.3  m sucrose , 2  mm mgoac2 , 1  mm cacl2 , 1% nonidet p-40 , 10  mm hepes , and ph  7.8 were then added to islk.rkshv219 cross - linked cells . the cells were scraped and placed within a 50  ml conical tube , and the nuclei were isolated by centrifugation at 1000  g for 5  min at 4  c .
micrococcal nuclease , mnase , is a internucleosomal cleavage reagent , which does not cleave the dna protected by a nucleosome .
islk.rkshv219 nuclei were digested with a titration of mnase : 4  u / ml , 2  u / ml , 1  u / ml , and 0.5  u / ml ( worthington biochemical corp . ) in mnase cleavage buffer ( 5  mm kcl , 4  mm mgcl2 , 1  mm cacl2 , 12.5% glycerol , and 50  mm tris  cl ( ph  7.4 ) ) .
a titration of mnase concentrations was utilized to ensure nucleosomes that are more and less readily cleaved are equally represented .
the nuclei , following mnase digestion , were treated with 0.2  g/l proteinase k and 1% sodium dodecyl sulfate .
mnase digested dna for all samples was resolved on a 2% agarose gel , and mononucleosomally protected dna , ~  150  bp , was isolated by gel excision .
following gel excision , the mononucleosomally protected dna was purified by electroelution , and for each sample 's mnase titration the mononucleosomally protected dna was combined .
the mononucleosomally protected dna was further purified with phenol  chloroform extraction and precipitated with alcohol .
islk.rkshv219 cells were resuspended in 0.2-g/l proteinase k and 1% sds solution and incubated at 55  c overnight for bare genomic dna isolation .
we utilized a high - resolution dna microarray , which we customized to contain 1  kb upstream and downstream of the transcription start sites of 462 immunity - related genes .
nimblegen 's 12-plex and hd2 design were utilized for the custom dna microarray 100319_hg19_immunity_jd_cgh array .
sequences were repeat - masked around the tsss , and only probes containing unique sequences for both the forward and reverse dna strands were printed on the microarray .
oligonucleotide probes , sized at 60  bp , were tiled on each subarray with a mean spacing of 47  bp overlap .
approximately 180 probes of spacing of 12.5  bp were printed on the microarray for each locus .
the samples were fluorescently labeled : nucleosomally protected dna with cy3 and bare genomic dna ( reference ) with cy5 .
samples were hybridized to the dna microarray according to nimblegen 's instructions ( http://www.nimblegen.com/technology/index.html ) .
the resulting .tiff files were used for microarray data feature extraction , primary data analysis and generation of initial data files using the nimblegen deva software version 1.2 cgh workflow .
specifically , loess spatial correction was applied to the raw data ; background correction was used to determine and adjust the signal data for noise using specifically designed background control probes ; and qspline normalization was applied to compensate for inherent differences in cy3 and cy5 signal .
the ratio between nucleosomal ( cy3 ) and bare genomic cy5 signals was then calculated and stored as a general feature format , .gff
subsequent analysis was performed using a software developed in our laboratory dennislab.r , which runs in the r environment for statistical computing .
dennislab.r was used to generate summary statistics on the range of nucleosome distribution values for each individual chromosome .
finally , we analyzed the nucleosome distribution changes at the 24  hour timepoint on a per chromosome basis , by calculating the t - statistic and p - value ( assuming unequal variance ) for the pairwise comparison of the range of values between all chromosomes .
in our previous work we demonstrated that nucleosome redistributions are widespread , transient , and dna - directed , .
we next wanted to determine if nucleosome redistributions showed chromosome specific changes in a time dependent manner .
we first calculated the range of nucleosome occupancy values for all loci studied on a per chromosome basis for each post - kshv reactivation timepoint ( fig . 
these results comport with our previous observation that the greatest positioning occurs at the 24  hour timepoint , however do not indicate chromosome specificity with respect to increased positioning .
we next wanted to investigate if the nucleosome redistribution changes at the 24  hour timepoint reflected increased or decreased nucleosomal occupancy .
we calculated the median nucleosome occupancy values on each chromosome for all of the loci measured .
we discovered that nucleosome occupancy values change in a chromosome specific manner ( fig . 
chromosomes 18 , 19 , and 22 show the greatest median nucleosome occupancy values at the 24  hour timepoint .
conversely , chromosomes 1 , 2 , 9 , 10 , 12 , 15 , and 17 show the lower median nucleosome occupancy values at the 24  hour timepoint .
we were next interested in calculating the statistical significance of the chromosome specific nucleosome occupancy changes that we had identified at the 24  hour timepoint .
most of the ranges of nucleosome occupancy values are not statistically significantly different from one another ( fig .  1c , yellow highlights ) .
these two chromosomes show clear increases in the nucleosome occupancy relative to the other chromosomes . at the other end of the scale , chromosomes 1 and 12 are statistically indistinguishable , yet different from all other chromosomes .
these results demonstrate that while nucleosomal positioning remains relatively constant between chromosomes at each timepoint , nucleosomal occupancy changes in a chromosome specific manner .
 in the eukaryotic nucleus , dna is packaged into chromatin . 
 the fundamental subunit of chromatin is the nucleosome , dna is wrapped 1.6 times around a histone octamer core . 
 nuclear processes in eukaryotes are impacted by whether regulatory dna is occupied by nucleosomes . 
 we used microarrays to measure nucleosome occupancy in human cells post - kaposi 's sarcoma - associated herpesvirus ( kshv ) reactivation at hundreds of immunity - related loci . 
 the detailed analysis of these technologies can be found in recent publications from our lab [ 1,3 ] . 
 we found that nucleosome redistributions displayed chromosome specific nucleosome occupancy . 
 this resource can be used to map nucleosome distributions in a variety of biological contexts . 
Direct link to deposited data
Experimental design, materials and methods
Cell culture and KSHV reactivation in iSLK.rKSHV219 cells
Post-KSHV reactivation cell harvest and nucleus purification
MNase digestion and purification of mononucleosomal protected DNA
Microarray design and processing
Data processing and analysis
Nucleosome occupancy changes occur in a chromosome specific manner