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, 2009). Surveying well-characterized genes with no known midline expression indicated that these genes had FPKM (Fragments Per Kilobase of exon per Million fragments mapped) values Adriamycin those expressed in the lateral CNS, sensory cells, midline accessory cells (channel glia, dorsal median cells), muscle, and trachea. Previous studies have characterized the CNS midline cell-type specific expression of 77 genes by confocal microscopy (Wheeler et al., 2009a?and?Wheeler et al., 2006). To initially validate the fidelity of the midline RNA-seq data, the FPKM values for these midline-expressed genes were assessed. Of the 77 genes, 75 are expressed between stages 11 and 17, the time-points used in our experiments. All 75 of these genes had an FPKM value >5 in either the 6�C8?h or 14�C16?h samples (or both) (Supplemental Table S4). Thus, we designate genes with a midline FPKM value ��5 as ��expressed�� in midline cells, and an FPKM value Pentamorphone abilities of the RNA-seq data, we addressed whether these data could identify factors that are enriched in the midline cells. To concentrate on transcription factors and transcriptional regulators, we first generated a list of 859 genes that contain the term ��transcription�� in their FlyBase gene ontology entries. We find that 508 of these genes are expressed in the 14�C16?h midline sample with FPKM values ��5 and 292 with FPKM values ��20. We then searched this list for genes with FPKM values ��20 in the 14�C16?h midline sample and LBH589 manufacturer that Drosophila cry encodes a blue light photoreceptor, and is not a transcription factor in Drosophila). Analysis in vivo by in situ hybridization confirmed that the expression of all 5 genes, in addition to sim, is highly enriched in the CNS midline ( Fig. 2C�CG). These genes were further analyzed for midline cell expression using fluorescent in situ hybridization in combination with midline cell type-specific markers ( Fig. 2B). The two circadian rhythm-associated genes, cry and per, show expression in non-overlapping midline neurons. Expression of the cry gene colocalizes with the MP1 neuron marker odd skipped (odd) ( Fig.