the facs - sorting strategy takes advantage of the ginip mouse model in which the mcherry fluorescent protein is expressed at ginip locus , allowing high fidelity identification of ginip neurons through live mcherry fluorescence . as shown in fig . 1 , combined analysis of ginip and ib4 expression segregates dorsal root ganglia ( drg ) neurons into 4 distinct subsets : ginip / ib4 double positive ( dp ) , ginip / ib4 , ginip / ib4 simple positive ( sp ) and ginip / ib4 double negative ( dn ) neurons . we have previously shown that dp neurons corresponded to the cutaneous mrgprd free nerve endings that convey noxious touch ,   and ginip / ib4 to the c - ltmrs , involved in pleasant touch , , . briefly , ginip drg cell suspensions were labeled with alexa647-conjugated ib4 and with sytox blue dye for exclusion of dying cells prior to facs sorting . drg cells suspensions from wild - type ( wt ) mice were used as a negative control . given that the final aim of this experiment is the extraction of high quality rna from selected neuronal subsets , the fine calibration of the facs experiment is a critical step . the basal fluorescence of drg cell suspensions in the mcherry detection channel was determined by plotting the structure ( ssc ) parameter versus mcherry parameter of sample from wt animals ( fig . this allowed reliable and specific detection of mcherry - positive cells within the ginip sample ( fig . the facs - gating strategy was as follows : we first defined ( a ) mcherry and ( b ) mcherry  sytox blue living cell gates . sp and dn subsets were sorted from gate a and dp and c - ltmrs were sorted from gate b ( fig . cells were sorted directly in rlt lysis buffer from rneasy micro kit ( quiagen ) and total rna was extracted according to manufacturer 's instructions . prior to rna - seq , the purity of the 4 sorted samples and the eventual cross contaminations were evaluated by rt - pcr . the expression patterns of ginip , mcherry , mrgprd , tafa4 , trpv1 and trka transcripts were analyzed and found accurate ( data not shown ) . however , sp cells were found to be highly contaminated by cd31-expressing endothelial cells that also bind ib4 and thus were excluded from the rna - seq experiment ( data not shown).the quality of the rna obtained from each sorted sample was determined with respect to rna profiles generated by the agilent bioanalyzer 2000 ( agilent inc . ) . for each sample , 50  ng of high quality total rnas were amplified using the amino allyl message amp ii amplification kit ( life technologies ) and 100  ng of resulting amplified rnas were used for libraries building with trueseq sample preparation ( low throughput protocol ) kit from illumina . libraries were sequenced in 100-bp paired - end cycles using the illumina hiseq2000 system with sbs technology . image analysis and base calling were performed using the hiseq control software and real - time analysis component provided by illumina . quality of the sequencing was assessed using fastqc and the illumina software sav ( sequence analysis viewer ) ; a representative example is shown in fig . 2 . for each sample , the median and the mean distribution of the quality scores was above 30 and data acquisition was not affected by base loss ( fig . b and data not shown ) . altogether , these criteria attest for the good quality of the rna - seq experiment . rna - seq reads were aligned to mus musculus genome ( ucsc mm10 ) using the eland_rna module from casava ( refflat.txt file downloaded from ucsc on october 10 , 2012 ) . as casava software is not designed for pair - end analysis , only the first pair was used to generate the counts . reads mapping to contaminants including 28s , 18s and 5s ribosomal rnas , mitochondrial chromosome , phix genome ( illumina control ) and illumina adaptors as well as reads mapping to multiple splicing positions and with no match , were removed . table 1 gives an overview of the number of counts generated and shows those that mapped to exons that were used for the creation of the raw data file . the first normalization method used was the reads per kilobase per million of mapped reads ( rpkm ) . analysis of rpkm values generated for genes expected to be enriched in each defined subset , showed substantial and accurate enrichment in mrgprd and p2rx3 in dp subset tafa4 and th in c - ltmrs and cgrp and sp in dp subset , providing a proof of concept of the rna - seq data presented in our study ( fig . 4a ) . because our aim was to determine the sets of differentially expressed genes ( degs ) in one subset as compared to the two others and that the rpkm normalization method was shown to be inappropriate for such an analysis , we chose the trimmed mean of m - values ( tmm ) normalization factors provided by edger package ( p  <  0,01 , fold change > smear - plot representation of pairwise comparison of tmm - normalized datasets showed that the log fold change ( fc ) was centered on zero , attesting that the libraries were correctly normalized ( see fig . 486 degs were declared enriched in dp as compared to the other subsets , 549 in c - ltmrs and 2916 in dn . only degs that generated at least 1000 reads in both biological replicates were kept for downstream data validation . as a result , data validation was carried on 156 , 184 and 784 dp- , c - ltmrs- and dn - deg , respectively . for this , we performed an extensive in situ hybridization ( ish ) screen on drg cryosections followed by either a ) ginip or ib4 immunostaining for dp and c - ltmrs datasets or b ) ginip and trka immunostaining for dn . the criteria for validation were the following : a ) a gene declared enriched in dp should be detected by ish as highly expressed in ginip / ib4 neurons but excluded from ginip / ib4 neurons ; a ) a gene declared enriched in c - ltmrs should exhibit the opposite expression pattern and c ) a gene declared enriched in dn should be excluded from ginip neurons . we thus monitored the expression of 48 dp- , 68-c - ltmrs and 13-dn - enriched transcripts thereby confirming the validity of rna - seq data generated in this study ( fig . moreover , we generated two open - access ish libraries ( dp ish library : http://www.ibdm.univ-mrs.fr/equipes/reynders/dp-ishlibrary.pptx and c - ltmrs ish library : http://www.ibdm.univ-mrs.fr/equipes/reynders/cltmrs-ishlibrary.pptx ) in which we provide the digital prevalence and the expression patterns of these genes in drg neurons . the facs - sorting strategy takes advantage of the ginip mouse model in which the mcherry fluorescent protein is expressed at ginip locus , allowing high fidelity identification of ginip neurons through live mcherry fluorescence . as shown in fig . 1 , combined analysis of ginip and ib4 expression segregates dorsal root ganglia ( drg ) neurons into 4 distinct subsets : ginip / ib4 double positive ( dp ) , ginip / ib4 , ginip / ib4 simple positive ( sp ) and ginip / ib4 double negative ( dn ) neurons . we have previously shown that dp neurons corresponded to the cutaneous mrgprd free nerve endings that convey noxious touch ,   and ginip / ib4 to the c - ltmrs , involved in pleasant touch , , . briefly , ginip drg cell suspensions were labeled with alexa647-conjugated ib4 and with sytox blue dye for exclusion of dying cells prior to facs sorting . drg cells suspensions from wild - type ( wt ) mice were used as a negative control . given that the final aim of this experiment is the extraction of high quality rna from selected neuronal subsets , the fine calibration of the facs experiment is a critical step . the basal fluorescence of drg cell suspensions in the mcherry detection channel was determined by plotting the structure ( ssc ) parameter versus mcherry parameter of sample from wt animals ( fig . this allowed reliable and specific detection of mcherry - positive cells within the ginip sample ( fig . the facs - gating strategy was as follows : we first defined ( a ) mcherry and ( b ) mcherry  sytox blue living cell gates . sp and dn subsets were sorted from gate a and dp and c - ltmrs were sorted from gate b ( fig . cells were sorted directly in rlt lysis buffer from rneasy micro kit ( quiagen ) and total rna was extracted according to manufacturer 's instructions . prior to rna - seq , the purity of the 4 sorted samples and the eventual cross contaminations were evaluated by rt - pcr . the expression patterns of ginip , mcherry , mrgprd , tafa4 , trpv1 and trka transcripts were analyzed and found accurate ( data not shown ) . however , sp cells were found to be highly contaminated by cd31-expressing endothelial cells that also bind ib4 and thus were excluded from the rna - seq experiment ( data not shown).the quality of the rna obtained from each sorted sample was determined with respect to rna profiles generated by the agilent bioanalyzer 2000 ( agilent inc . ) . for each sample , 50  ng of high quality total rnas were amplified using the amino allyl message amp ii amplification kit ( life technologies ) and 100  ng of resulting amplified rnas were used for libraries building with trueseq sample preparation ( low throughput protocol ) kit from illumina . libraries were sequenced in 100-bp paired - end cycles using the illumina hiseq2000 system with sbs technology . image analysis and base calling were performed using the hiseq control software and real - time analysis component provided by illumina . quality of the sequencing was assessed using fastqc and the illumina software sav ( sequence analysis viewer ) ; a representative example is shown in fig . the median and the mean distribution of the quality scores was above 30 and data acquisition was not affected by base loss ( fig . 3a altogether , these criteria attest for the good quality of the rna - seq experiment . rna - seq reads were aligned to mus musculus genome ( ucsc mm10 ) using the eland_rna module from casava ( refflat.txt file downloaded from ucsc on october 10 , 2012 ) . as casava software is not designed for pair - end analysis , only the first pair was used to generate the counts . reads mapping to contaminants including 28s , 18s and 5s ribosomal rnas , mitochondrial chromosome , phix genome ( illumina control ) and illumina adaptors as well as reads mapping to multiple splicing positions and with no match , were removed . table 1 gives an overview of the number of counts generated and shows those that mapped to exons that were used for the creation of the raw data file . the first normalization method used was the reads per kilobase per million of mapped reads ( rpkm ) . analysis of rpkm values generated for genes expected to be enriched in each defined subset , showed substantial and accurate enrichment in mrgprd and p2rx3 in dp subset tafa4 and th in c - ltmrs and cgrp and sp in dp subset , providing a proof of concept of the rna - seq data presented in our study ( fig . 4a ) . because our aim was to determine the sets of differentially expressed genes ( degs ) in one subset as compared to the two others and that the rpkm normalization method was shown to be inappropriate for such an analysis , we chose the trimmed mean of m - values ( tmm ) normalization factors provided by edger package ( p  <  0,01 , fold change > smear - plot representation of pairwise comparison of tmm - normalized datasets showed that the log fold change ( fc ) was centered on zero , attesting that the libraries were correctly normalized ( see fig . 486 degs were declared enriched in dp as compared to the other subsets , 549 in c - ltmrs and 2916 in dn . only degs that generated at least 1000 reads in both biological replicates were kept for downstream data validation . as a result , data validation was carried on 156 , 184 and 784 dp- , c - ltmrs- and dn - deg , respectively . for this , we performed an extensive in situ hybridization ( ish ) screen on drg cryosections followed by either a ) ginip or ib4 immunostaining for dp and c - ltmrs datasets or b ) ginip and trka immunostaining for dn . the criteria for validation were the following : a ) a gene declared enriched in dp should be detected by ish as highly expressed in ginip / ib4 neurons but excluded from ginip / ib4 neurons ; a ) a gene declared enriched in c - ltmrs should exhibit the opposite expression pattern and c ) a gene declared enriched in dn should be excluded from ginip neurons . we thus monitored the expression of 48 dp- , 68-c - ltmrs and 13-dn - enriched transcripts thereby confirming the validity of rna - seq data generated in this study ( fig . moreover , we generated two open - access ish libraries ( dp ish library : http://www.ibdm.univ-mrs.fr/equipes/reynders/dp-ishlibrary.pptx and c - ltmrs ish library : http://www.ibdm.univ-mrs.fr/equipes/reynders/cltmrs-ishlibrary.pptx ) in which we provide the digital prevalence and the expression patterns of these genes in drg neurons . we described here a unique dataset encompassing the combinatorial gene expression enriched in mrgprd , c - ltmrs and dn drg sensory neurons . we highlight here that our published rna - seq data are of good quality and concordant with our validation strategy . these data provide a wealth of information regarding the presence and the prevalence of transcripts in mrgprd , c - ltmrs and dn sensory neurons , they highlight the differential gene expression in two functionally opposite classes of cutaneous afferents and they allow open - access monitoring the cellular distribution of over 100s of genes .
the skin is the largest sensory organ that is densely innervated by highly specialized sensory neurons allowing the detection of a wide range of stimulations including light touch , temperature , itch and pain . 
 our knowledge of the sets of genes instructing the functional specialization of sensory neurons is just emerging . in a previous study , we have identified a new gi inhibitory interacting protein ( ginip ) that marks two distinct subsets of skin - innervating sensory neurons conveying noxious and pleasant touch : the mrgprd - expressing c - fibers specialized in noxious touch and the th+/tafa4+/v - glut3 + c - low threshold mechanoreceptors ( c - ltmrs ) , part of neurons processing pleasant touch . in the recent study published by reynders et al . 
 ( 2015 ) , we took advantage of ginipmcherry mouse model in combination with isolectin b4 ( ib4 ) cell surface labeling and fluorescence activated cell sorting ( facs ) . 
 we successfully purified mrgprd+ , c - ltmrs and a heterogeneous population of sensory neurons and subjected their rna contents rna - deep sequencing ( rna - seq ) . 
 the subsequent rna - seq experiment led to the generation of unique sets of data representative of pure transcriptome profiles of each subset . as a result of this pioneering approach , 
 we established the combinatorial expression of the sets of genes that could dictate the functional specializations of mrgprd+ neurons and c - ltmrs . herein we provide details regarding the experimental design , the quality controls and statistical analysis of the data deposited at gene expression omnibus under the accession number gse64091 .