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to specifically delete mature osteolineage cells , the oc - cre strain ( from dr .
thomas l. clemens , johns hopkins university , md , usa ) , which expressed cre under the osteocalcin promoter was crossed to the idtr strain .
temporally controlled cell ablation was achieved upon injection of diphtheria toxin into the ocncre;idtr strain . to study
the effect of ocn  cells on hematopoiesis , ocncre injected with diphtheria toxin or ocncre;idtr injected with pbs served as controls while ocncre;idtr injected with diphtheria toxin were used as mutants . for most experiments , 25  ng diphtheria toxin in pbs / g of body weight
was injected daily into both control and mutant animals for 28  days from 4  weeks of age to achieve an acute deletion of specific osteolineage populations .
mice were harvested for analysis the next day after the last dose of diphtheria injection .
for all experiments , littermates were used as controls . all animal usage and procedures performed
to specifically delete mature osteolineage cells , the oc - cre strain ( from dr .
thomas l. clemens , johns hopkins university , md , usa ) , which expressed cre under the osteocalcin promoter was crossed to the idtr strain .
temporally controlled cell ablation was achieved upon injection of diphtheria toxin into the ocncre;idtr strain . to study
the effect of ocn  cells on hematopoiesis , ocncre injected with diphtheria toxin or ocncre;idtr injected with pbs served as controls while ocncre;idtr injected with diphtheria toxin were used as mutants . for most experiments , 25  ng diphtheria toxin in pbs / g of body weight
was injected daily into both control and mutant animals for 28  days from 4  weeks of age to achieve an acute deletion of specific osteolineage populations .
mice were harvested for analysis the next day after the last dose of diphtheria injection .
for all experiments , littermates were used as controls . all animal usage and procedures performed
before sacrifice , peripheral blood was collected from each mouse and subjected to complete blood cell count . for each mouse ,
tibiae , femurs , iliac crests , spines , ulnae , radii , and humeri were collected for bone marrow cells .
we routinely stain 5    10 cells per sample for the hematopoietic stem population , and 1    10 cells per sample for each progenitor and mature population .
lineage cocktail consists of biotinylated b220 , cd3e , cd4 , cd8a , cd19 , cd11b , gr1 , ter119 , cd11c , and nk1.1 antibodies .
the following antibody combinations were used to recognize ly6d clp ( lineage - pacific orange , sca - pacific blue , ckit - apc - cy7 , cd127-pe - cy7 , thy1.2-fitc , ly6d - apc ) .
ly6d clps were sorted from 6 to 8  weeks old ocncre;idtr mutants and controls using the facsaria following daily dt treatment for 4  weeks .
rna was extracted using the trizol ( invitrogen ) according to the manufacturer 's instructions .
samples were processed by the nugen ovation v2 laboratory process in the microarray core facility of dana - farber cancer institute .
briefly , first stand cdna was prepared from total rna using a dna / rna chimeric primer and a reverse transcriptase .
the resulting double stranded cdna with a unique heteroduplex at the 5 ' end of the antisense strand were amplified using spiatm amplification , a repeated process of spiatm dna / rna primer hybridization , dna replication , strand displacement and rna cleavage which resulted in a rapid accumulation of cdna with sequence complementary to the original rna .
the spiatm amplified cdna was purified using the zymo research dna clean & concentrator system .
the purified cdna was fragmented through a chemical and enzymatic process and labeled via enzymatic attachment of a biotin - labeled nucleotide to the 3-hydroxyl end of the fragmented cdna .
the biotinylated cdna was added to a hybridization solution containing several biotinylated control oligonucleotides ( for quality control ) , and hybridized to the mouse430a microarray chip overnight at 45  c .
the chips were then transferred to a fluidics instrument that performs washes to remove cdna that has not hybridized to its complementary oligonucleotide probe .
the bound cdna was then fluorescently labeled using phycoerythrin - conjugated streptavidin ( sape ) ; additional fluors were then added using biotinylated anti - streptavidin antibody and additional sape .
each cdna bound at its complementary oligonucleotide was excited using a confocal laser scanner , and the positions and intensities of the fluorescent emissions were captured .
standard qa / qc analyses involved chip analysis with the assayqualitymetrics bioconductor package and found no significant quality issues with any of the chips , as determined by ( among other methods ) visual inspection , intensity distributions or rna degradation plots .
the data was background corrected and normalized with rma ( robust multichip average ) using the  affy  bioconductor package .
the selected database comprises 6 expression measurements of 41,345 genes , 3 in the first condition and 3 in the second .
the recorded expression values ranged between 4.023 and 10,198.29 . supervised learning or class prediction methods were used for molecular classification and pattern recognition .
this analysis involves selecting the features ( genes ) most correlated with a phenotypic distinction of interest . these features or  marker genes 
are biologically interesting in themselves but they can also be used as the input of a classification algorithm that uses existing  labeled  samples to build a model to predict the labels for future samples .
genes correlated with a binary class distinction , for example a morphological or clinical phenotype , is directly identified and selected by using a  distance  metric , for example t - test statistic  =  ( ma    mb )    ( s2a  +  s2b ) [ m and s are the means and std .
dev . per class ] or signal to noise ratio  =  ( ma    mb )  /  ( sa  +  sb ) [ m and s are the means and std . dev . per class ] .
1 is a color map illustrating 30 genes overexpressed in mutant ly6d clps compared to control ly6d clps .
table 1 shows the mean , standard deviation , score , and fold change of the 30 genes .
t cells are known to develop in the thymus . 
 however , molecular events that control the transition from hematopoietic progenitor cells in the bone marrow to t precursor cells seeded in the thymus remained poorly defined . 
 our recent report showed that osteocalcin ( ocn)-expressing bone cells in the bone marrow have major impact on t cell immunity by regulating t progenitor development in the bone marrow ( yu et al . , 2015 ) [ 1 ] . 
 selective endogenous depletion of ocn+  cells by inducible diphtheria toxin receptor expression ( ocncre;idtr ) led to reduction of t - competent common lymphoid progenitors ( ly6d clps ) in the bone marrow and loss of t cells in the thymus . 
 expression of the notch ligand dll4 by ocn+  cells in the bone marrow ensures the production of ly6d clps , and expression of chemotactic molecules ccr7 and psgl1 to enable subsequent thymic seeding . 
 these data indicate that specific mesenchymal cells in bone marrow provide key molecular drivers enforcing thymus - seeding progenitor generation and thereby directly link skeletal biology to the production of t cell based adaptive immunity . here 
 we present the transcriptome profiles of ly6d clps derived from ocn+ cells deleted mice ( ocncre+;idtr ) compared to those derived from control littermates ( ocncre;idtr ) . 
 these data are publically available from ncbi gene expression omnibus ( geo ) with the accession number gse66102 .