![]() ![]() Many exploit dyes or viruses that are injected into the target structure, internalized at axon terminals, and trafficked retrogradely to label cell bodies in the source structure ( Wickersham and Feinberg, 2012). Therefore, in recent years, several approaches have been developed to focus transcriptional profiling on projection populations of interest. This is a powerful, systematic approach, but entails obtaining or generating Cre transgenic mice for each population and is mouse and labor intensive. One approach to this correspondence problem is to sequence a tissue, identify markers for many different cell types, and perform viral anterograde tracing in a panel of transgenic mice expressing Cre recombinase ( Ding et al., 2020). However, a challenge in interpreting single-cell RNA-seq datasets is to link populations identified through RNA expression to anatomy, connectivity, and circuit properties, a conundrum known as a “correspondence problem” ( Lein et al., 2017). Single-cell RNA sequencing (RNA-seq) technologies offer insights into the physiology of each identified neuronal population and molecular markers that could be used for targeted monitoring and manipulations during behavior. A central goal of neuroscience is deciphering the properties and behavioral functions of the myriad projection neuron subtypes in the brain. For example, primary visual cortex contains functionally distinct populations that project to higher visual cortical areas, contralateral cortex, and subcortical targets such as striatum, thalamus, and superior colliculus and substantia nigra pars reticulata in midbrain harbors subtypes that project to 39 target structures and differ in neurotransmitters released, response tuning, and intrinsic excitability ( Antal et al., 2014 Jiang et al., 2003 Kim et al., 2015 Lur et al., 2016 McElvain et al., 2021 Poulin et al., 2016). Our study provides a roadmap for high-throughput identification of neuronal subtypes based on connectivity.įunctionally and molecularly diverse projection neurons with distinct targets are intermingled in most brain areas. VECTORseq is compatible with different viral families, resolves multiple populations with different projection targets in one sequencing run, and identifies cortical and subcortical excitatory and inhibitory projection populations. VECTORseq repurposes commercial retrogradely infecting viruses typically used to express functional transgenes (e.g., recombinases and fluorescent proteins) by treating viral transgene mRNA as barcodes within single-cell datasets. We developed a straightforward, multiplexed approach, virally encoded connectivity transgenic overlay RNA sequencing (VECTORseq). Single-cell RNA sequencing has facilitated these efforts by revealing molecular determinants of cellular physiology and markers that enable genetically targeted perturbations such as optogenetics, but existing methods for sequencing defined projection populations are low throughput, painstaking, and costly. ![]() ![]() Consequently, a major focus of modern neuroscience is defining the physiology and behavioral roles of projection neurons linking different brain areas. Quality control prices for more than one sample (if required) apply.Behavior arises from concerted activity throughout the brain. MiSeq sequencing service includes instrument operation by technician and the charge for MiSeq use per hour. The service prices include charges per hour of MiSeq run, sequencing by technician, and sample quality control if required by the customer. For a MiSeq run, the customer should order and bring own Illumina sequencing kit that fits length and type requirements of the specific experiment. In addition to high-throughput Illumina HiSeq sequencing in High-Output mode (150-200 million reads per lane) and Rapid mode (100-120 million reads per lane), we provide a lower-throughput but fast and less expensive MiSeq sequencing (10-15 million reads per run). Prices for various types of library construction are also given below, and include validation of input DNA or RNA, library construction, and library validation. Prices for quality control of libraries after the first are given below. Price per lane of sequencing includes quality control of one library (or total of multiple librares if pooled by the customer), cluster generation, sequencing, demultiplexing of standard Illumina barcode reads, and online delivery of sequence and quality data. ![]() Please contact us for specific pricing information on Bioinformatics consulting. Additional fees apply for downstream Bioinformatics analysis services or consultations. Basic pricing for NextGen library construction and sequencing services is given below. ![]()
0 Comments
Leave a Reply. |
AuthorWrite something about yourself. No need to be fancy, just an overview. ArchivesCategories |