Next Generation Sequencing Investigation of altered transcripts in presence of dominant-negative transcription factor

Purpose:The goals of this study was to determine alterations in expression levels of transcripts downstream of a dominant-negative transcription factor. Quantitative reverse transcription polymerase chain reaction (qRT–PCR) methods was used to confirm the altered expression of targets. Methods: Striatal mRNA profiles of 11-month-old wild-type (WT) and Nestin-Cre X PPAR delta E411P mice were generated by deep sequencing, in triplicate, using Illumina HiSeq 2000. The sequence reads that passed quality filters were analyzed at the transcript isoform level with TopHat followed by Cufflinks. qRT–PCR validation was performed using TaqMan and SYBR Green assays. Western blots, and immunofluorescence was also used to confirm if altered mRNA levels translated to changes at the protein level. Results: Using data analysis workflow, we mapped sequence reads for each sample to the mouse genome (build mm9) and identified transcripts in the striatum of WT and PPARdelta E411P mice. Conclusions: Our study found multiple transcripts altered in the striatum of the Nestin-Cre x PPAR delta E411P mice as compared to WT striatum, as generated by RNA-SEQ in biologic replicates. Overall design: Striatal mRNA profiles of 11-month-old wild type (WT) and Nestin-Cre X PPAR delta E411P mice were generated by deep sequencing, in triplicate, using Illumina HiSeq2000.

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Dickey AS, Pineda VV, Tsunemi T, Liu PP, Miranda HC, Gilmore-Hall SK, Lomas N, Sampat KR, Buttgereit A, Torres MJ, Flores AL, Arreola M, Arbez N, Akimov SS, Gaasterland T, Lazarowski ER, Ross CA, Yeo GW, Sopher BL, Magnuson GK, Pinkerton AB, Masliah E, La Spada AR