The sequencing core receives 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 are be processed by the core. The Sequencing Core receives spleens from Research Projects 1-3 that will be used for genotyping. Genotype data is produced using genotyping by sequencing (GBS), which is an innovative technology that takes advantage of the rapid advances in next-generation sequencing. This core extracts DNA from the spleens, makes GBS libraries, multiplexes those libraries, and sequences them using an Illumina 4000 equipment. The raw sequencing reads are sent to Research Project 4 for further analysis.
The Sequencing Core also performs RNA sequencing (RNAseq) of 5 key brain regions: nucleus accumbens core (AcbC), lateral habenula (LHb), prelimbic cortex (PC), infralimic cortex (IL) and orbitofrontal cortex (OFC). Total RNA from these brain regions is supplied by Research Project 2, which will use microdissection to accurately dissect these regions from HS rats. This core prepare libraries, multiplex them at a density of 12-15 per lane and sequence from a single end, 100 bp, using an Illumina 4000, to obtain 25-30 million reads per library. 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 has access IGM Core at UCSD as well as next-generation sequencing machines from other companies.
This core may also 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.