Gene expression difference between normal and Pten-deficient 5-HT neurons

Purpose: compare the gene expression diffenrence between wildtype and Pten-deficient 5-HT neurons. Methods: 5-HT neurons cell body are collected from acut brain slice with patch electrodes. Pten-deficient 5-HT neurons are obtained from Pet1-Cre; Ptenflox/flox mice. Normal 5-HT neurons are obtained from littermate wildtype mice. Reverse transcription was performed using a SMARTer Ultra Low RNA Kit (Clontech, Japan). Single-cell cDNA was amplified using an Advantage 2 PCR Kit (Clontech, Japan). Purified cDNA from individual cells were sequenced using the Illumina HiSeq 2000 platform. Sequence reads were aligned to the mouse mm9 genome using TopHat. Differential expression analysis was performed using DESeq (Bioconductor) followed by Student t-test. Genes were considered differentially expressed if p<0.05. Results: The somatic compartments of individual YFP-labeled 5-HT neurons in acute brain slices were harvested for RNA sequencing. Each of the cells had 2 X 107 reads mapping back to the mouse genome when analyzed using the RUM mapping program. We found 859 genes (381 up-regulated, 478 down-regulated) were differentially expressed in Pten-deficient 5-HT neurons. Gene ontology of differentially expressed genes revealed the biological processes up-regulated and down-regulated in Pten-deficient 5-HT neurons. The absence of Pten in 5-HT neurons was associated with significantly increased expression of genes involved in the regulation of axonal extension and cell size, cell morphogenesis and cytoskeleton organization mRNA profile and comparison between wildtype and Pten-deficient 5-HT neurons.