(Core C: Sequencing Core) The sequencing core will receive tissue samples from Research Projects 1-3 and use next-generation sequence to produce data that will be analyzed by Research Project 4. Two types of tissue samples will be processed by the core. First, the Sequencing Core will receive spleens from Research Projects 1-3 that will be used for genotyping. Genotype data will be produced using genotype by sequencing (GBS), which is an innovative technology that takes advantage of the rapid advances in next-generation sequencing. This core will extract DNA from the spleens, make GBS libraries, multiplex those libraries at a density of 12 per lane, and sequence them using an Illumina HiSeq 2500. The raw sequencing reads will be sent to Research Project 4 for further analysis. Second, the Sequencing Core will also perform RNA sequencing (RNAseq) of 4 key brain regions: the nucleus accumbens core (AcbC), the lateral habenula (LHb), the infralimic cortex (IL) and the orbitofrontal cortex (OFC). Total RNA from these brain regions will be supplied by Research Project 2, which will use laser capture microdissection (LCM) to accurately dissect these regions from HS rats. This core will perform an amplification step prior to preparing libraries because the amount of mRNA obtained via LCM will be relatively small. We will multiplex those libraries at a density of 5 per lane and sequence them using an Illumina HiSeq 2500. The raw reads will be passed to Research Project 4 for further analysis. The PI's laboratory (Dr. Palmer) has extensive experience with all necessary techniques. This core will have access to numerous next-generation sequencing machines that are already in place at the University of Chicago. In particular, the Department of Human Genetics maintains an Illumina HiSeq 2500;this core will only have to pay for the cost of reagents to use that machine. Dr. Palmer also has access to at least 5 other HiSeq 2500 machines at the University of Chicago, as well as next-generation sequencing machines from other companies (e.g. 454, Pacific Biosystems, etc.). Additional machines are also available at other participating institutions. Because sequencing technology will continue to evolve rapidly, we anticipate that some of the technical details that we describe will change over the proposed funding period;since those changes are difficult to predict we have focused on describing technologies that are currently in use in Dr. Palmer's lab. Note that we are not proposing to purchase any major equipment (e.g. we are not proposing to buy a next- generation sequencing machine) as part of this core. This allows us to deliver the necessary services while minimizing the costs paid by the center. We also discuss the possibility that this core would provide GBS services to outside investigators on a cost recovery basis. Because the analysis of GBS data requires large numbers of samples, such a service would only be appropriate for investigators who are interested in genotyping at least several hundred samples.
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