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peripheral blood monocytes were isolated from three buffy coats ( blutspende zrich , zurich , switzerland ) by density gradient centrifugation ( ficoll - paque plus ( ge healthcare ) ) and enriched for co - culture assays using human monocyte isolation kit ii ( macs , miltenyi biotech ) .
the breast cancer cell lines mda - mb-231 and t47d cells ( kindly provided by dr .
nancy e. hynes , fmi , basel , switzerland ) were maintained in dmem ( gibco ) supplemented with 10% fetal bovine serum ( sigma ) and penicillin / streptomycin .
the breast cancer cell lines were plated 1    10 cells in the lower compartment of 75  mm polycarbonate transwell inserts pore size 0.4  m ( corning ) in maintenance medium one day prior monocyte isolation . the next day
the medium was replaced by rpmi ( gibco ) on hour before the addition of 4    10 monocytes into the upper compartment of the transwell inserts .
the cells were co - cultured for five days in a humidified chamber at 37  c .
control wells contained either cancer cells only in the lower compartment with rpmi in the upper compartment or rpmi in the lower compartment with only monocytes in the upper compartment .
the total rna from 17 samples was isolated using the trizol protocol ( invitrogen ) .
the rna quantity was determined by nanodrop spectrophotometer and quality assessed on the agilent 2100 bioanalyzer ( agilent ) using an rna 6000 nano chip .
two samples , donor2 monocytes cultured as single culture ( m2c ) and with t47d co - culture ( m2 t ) did not reach sufficient amount of rna and were excluded from further processing . at this point the samples were sent to bgi china where they were processed according to bgi 's standard rna - seq sample preparation .
shortly , magnetic beads with oligo ( dt ) were used to isolate mrna , which was mixed with the fragmentation buffer to fragment it into short fragments .
the cleaved rna fragments were synthesized into single - strand cdna using superscript ii reverse transcriptase and random hexamers followed by second strand synthesis with dna polymerase i and escherichia coli rnase h. after the second strand synthesis , with end repair and a - tailing , the synthesized double - stranded cdna fragments were subjected to purification , ligated to illumina adapters using quick ligation tm kit ( neb ) and dna ligase .
the resultant cdna adapter - modified cdna libraries were fractionated on agarose gel , 200-bp fragments were excised and amplified by 15  cycles of polymerase chain reaction .
after purification the quality of cdna libraries was checked by bioanalyzer 2100 ( agilent ) .
the concentration of the cdna libraries was measured and diluted to 10  nm in tris 
cluster formation , primer hybridization and sequencing reactions were performed sequentially according to the manufacturer 's recommended protocol . in the present study , we used pair - end sequencing by illumina hiseqtm 2000 ( illumina , san diego , ca , usa ) .
high - quality reads were aligned to the human reference genome with soapaligner / soap2 .
the expression level for each gene was normalized to reads per kilobase per million mapped reads ( rpkm ) to facilitate the comparison of transcripts among samples .
a mean log2 ratio [ rpkm of monocytes co - cultured with mda - mb-231 or t47d cells / monocytes cultured alone ; rpkm of mda - mb231 or t47d cells co - cultured with monocytes / mda - mb231 or t47d cells cultured alone ] of each gene was calculated . to identify genes differentially expressed between groups we used an algorithm based on ref .   with a correction for false positive ( type i ) and false negative ( type ii ) errors using the fdr method .
further , genes were classified as up regulated when their mean log2 ratio was larger than 0.5 or down regulated when their log2 ratio was less than   0.5 .
peripheral blood monocytes were isolated from three buffy coats ( blutspende zrich , zurich , switzerland ) by density gradient centrifugation ( ficoll - paque plus ( ge healthcare ) ) and enriched for co - culture assays using human monocyte isolation kit ii ( macs , miltenyi biotech ) .
the breast cancer cell lines mda - mb-231 and t47d cells ( kindly provided by dr .
nancy e. hynes , fmi , basel , switzerland ) were maintained in dmem ( gibco ) supplemented with 10% fetal bovine serum ( sigma ) and penicillin / streptomycin .
the breast cancer cell lines were plated 1    10 cells in the lower compartment of 75  mm polycarbonate transwell inserts pore size 0.4  m ( corning ) in maintenance medium one day prior monocyte isolation . the next day
the medium was replaced by rpmi ( gibco ) on hour before the addition of 4    10 monocytes into the upper compartment of the transwell inserts .
the cells were co - cultured for five days in a humidified chamber at 37  c .
control wells contained either cancer cells only in the lower compartment with rpmi in the upper compartment or rpmi in the lower compartment with only monocytes in the upper compartment .
the total rna from 17 samples was isolated using the trizol protocol ( invitrogen ) . see table 1 for sample identity .
the rna quantity was determined by nanodrop spectrophotometer and quality assessed on the agilent 2100 bioanalyzer ( agilent ) using an rna 6000 nano chip .
two samples , donor2 monocytes cultured as single culture ( m2c ) and with t47d co - culture ( m2 t ) did not reach sufficient amount of rna and were excluded from further processing . at this point the samples were sent to bgi china where they were processed according to bgi 's standard rna - seq sample preparation . shortly , magnetic beads with oligo ( dt ) were used to isolate mrna , which was mixed with the fragmentation buffer to fragment it into short fragments .
the cleaved rna fragments were synthesized into single - strand cdna using superscript ii reverse transcriptase and random hexamers followed by second strand synthesis with dna polymerase i and escherichia coli rnase h. after the second strand synthesis , with end repair and a - tailing , the synthesized double - stranded cdna fragments were subjected to purification , ligated to illumina adapters using quick ligation tm kit ( neb ) and dna ligase .
the resultant cdna adapter - modified cdna libraries were fractionated on agarose gel , 200-bp fragments were excised and amplified by 15  cycles of polymerase chain reaction .
after purification the quality of cdna libraries was checked by bioanalyzer 2100 ( agilent ) .
the concentration of the cdna libraries was measured and diluted to 10  nm in tris 
cluster formation , primer hybridization and sequencing reactions were performed sequentially according to the manufacturer 's recommended protocol . in the present study
, we used pair - end sequencing by illumina hiseqtm 2000 ( illumina , san diego , ca , usa ) .
high - quality reads were aligned to the human reference genome with soapaligner / soap2 .
the expression level for each gene was normalized to reads per kilobase per million mapped reads ( rpkm ) to facilitate the comparison of transcripts among samples . a mean log2 ratio [ rpkm of monocytes co - cultured with mda - mb-231 or t47d cells / monocytes cultured alone ; rpkm of mda - mb231 or t47d cells co - cultured with monocytes / mda - mb231 or t47d cells cultured alone ] of each gene was calculated . to identify genes differentially expressed between groups we used an algorithm based on ref .   with a correction for false positive ( type i ) and false negative ( type ii ) errors using the fdr method
further , genes were classified as up regulated when their mean log2 ratio was larger than 0.5 or down regulated when their log2 ratio was less than   0.5 .
we describe here a dataset composed of rna - seq gene expression profiling of macrophage - cancer cell crosstalk using two different breast cancer cell lines with distinct aggressive phenotype . with this data
we could show that macrophage response to different cancer cells does not necessarily promote a tumor - supporting inflammatory response since a pro - inflammatory gene signature was activated in macrophages co - cultured with estrogen positive breast cancer cells .
macrophages are important mediators of tumor progression and their function is broadly influenced by different microenvironmental stimuli . to understand the molecular basis of the tumor - supporting role of macrophages in aggressive breast cancer we co - cultured human peripheral monocytes with two breast cancer cell lines representing distinct aggressive cellular phenotype and transcriptionally profiled the changes occurring in both cells during in vitro activated crosstalk . 
 here we provide a detailed description of the experimental design , sample identity and analysis of the illumina rna - seq data , which have been deposited into gene expression omnibus ( geo ) : gse75130 .