Dr. Peter Adams, Sanford Burnham Prebys Medical Discovery Institute
Dr. Geoffrey Wahl, Salk Institute for Biological Studies
Dr. Brian James, Sanford Burnham Prebys Medical Discovery Institute
Single Cell Sequencing Workshop Group/Social
Date: First Monday Monthly
Time: 5:00pm - 6:00pm
Sanford Burnham Prebys Medical Discovery Institute
Fishman Auditorium, Building #4
10901 North Torrey Pines Road
La Jolla, CA 92037
2017 Workshop Group/Social Dates
Monday, March 6, 2017
Monday, April 3, 2017
Monday, May 1, 2017
Monday, June 5, 2017:
Presenter: Brandon Sos from Kun Zhang’s lab at UCSD; Title: The Single Cell Transposome Hypersensitive Sites Sequencing (scTHS-seq) assay for Chromatin Accessibility and Assessment of Epigenetic States in the Human Adult Brain;
Summary: Chromatin accessibility captures in vivo protein-chromosome binding status, and is considered an informative proxy for protein-DNA interactions. Current single cell chromatin accessibility assays result in sparse chromatin maps at high throughput or dense chromatin maps at low throughput. We present a high throughput transposome hypersensitive sites sequencing assay for highly sensitive characterization of chromatin accessibility in single cells, generating thousands of single cell datasets from the human adult visual and frontal cortex. Integrative analysis of transcriptomic snDrop-seq data and scTHS-seq data has allowed us to identify transcription factors and regulatory elements shaping the state of different brain cell types, and to map genetic risk factors of common human brain diseases to specific pathogenic cell types and subtypes.
Monday, August 7, 2017:
Presenter: Chungyuan Luo from Dr. Joseph Ecker Lab at SALK; Title: Single Neuronal Methylomes Reveal Epigenomic Diversity in the Mammalian Brain
Summary: Epigenomic marks such as cytosine DNA methylation (mC) have highly diverse patterns across brain neuronal and non-neuronal cell types. Human and mouse brains accumulate high levels of non-CG methylation (mCH) at locations throughout the genome that are inversely correlated with gene expression. In addition, more than 200,000 regions showing differential CG methylation were identified between three cortical excitatory and inhibitory neuron types. Approximately 16% of the mouse genome contains differential mC signatures that allows neuronal cell types to be distinguished by low coverage single cell WGBS (whole-genome bisulfite sequencing). Extending cell type specific mC analysis to all (known and unknown) brain cell types requires unbiased single cell mC profiling, which will also enables the study of mC heterogeneity across cells of the same type.To meet the need of large-scale single cell methylome profiling, we developed a new method for the preparation of single cell methylome libraries using Swift Biosciences AdaptaseTM technology. Our strategy generates libraries with greater complexity than commercially available methods and allows the pooling of single cells for high-throughput library preparation. Single cell methylomes were generated from over 6,000 single neuronal nuclei isolated from human and mouse frontal cortex using FACS. We demonstrated robust cell type classification using single cell methylomes, readily separating excitatory and inhibitory populations and also identifying distinct neuronal subtypes. The single cell methylome method will enable unbiased characterization of brain epigenomic diversity without the need of the isolation of specific cell populations.
Monday, October 2, 2017:
Presenter: Dr. Zhuzhu Zhang from Dr. Joseph Ecker Lab at SALK;
Title: Single-cell methylome sequencing of identified cortical projection cell types
Summary: Unlike in most parts of the body where DNA methylation is mature at birth, in the mammalian cerebral cortex DNA methylation increases over a protracted postnatal time period which mirrors the developmental timing of an expansion and then subsequent pruning of cortical connections. For example, individual neurons in the adult mouse primary visual cortex (V1) project to just one of nine adjacent visual cortical areas. This arrangement emerges from an initial configuration in which each V1 neuron projects to multiple areas. The subsequent rearrangement occurs through postnatal axonal pruning at the same time as the increase in DNA methylation. These observations suggest that postnatal epigenetic mechanisms play a role in matching gene expression to connectivity. This hypothesis predicts that V1 cells projecting to different visual cortical areas will differ in gene expression and methylation. To investigate our hypothesis, we injected retrogradely-infecting virus that expresses Cre-recombinase into two distinct higher visual cortical areas of INTACT mice that have Cre-dependent expression of a nuclear envelope anchored GFP protein. Single nuclei of retrogradely infected cortico-cortical subtypes within V1 were subsequently isolated by fluorescence-activated cell sorting (FACS). We then performed single-cell methylome sequencing to characterize and compare their genome-wide DNA methylation profiles. Our preliminary results suggest that these two groups of V1 cortico-cortical neurons have different non-CG methylation patterns. Such a correspondence between DNA methylation and projection target suggests that epigenetic mechanisms work in concert with activity and experience-dependent rearrangements of connectivity to establish appropriate matching between genetically determined functional specializations and environmentally imposed responses to sensory stimuli.
Presenter: Marco Maruggi, M. Eng. (PhD Candidate, NIH NRSA Predoctoral Fellow) from Dr. Garth Powis Lab at Sanford Burnham Prebys Medical Discovery Institute;
Title: Characterization of VEGF-Independent Angiogenesis in a HIF1A-Knockdown Model
Summary: HIF-1 is a transcription factor regulating the production of the vascular endothelial growth factor (VEGF) and is essential for the growth of solid tumors as a response to hypoxia due to poor tumor blood supply. We have found that a pancreatic cancer cell line where HIF-1 is inhibited does not immediately form tumors when implanted in mice, but with time an aggressive HIF-1 independent tumor grows out. Determining what allows these tumors to grow without HIF-1 has significant implications in understanding the development of resistance in patients to anti-VEGF therapy such as Avastin, and it could also provide clues why Avastin therapy of patients with macular degeneration is successful in only a subset of individuals. Using bulk RNA analysis we have identified a number of immoattractant cytokines secreted by the tumor, and an increased immune infiltration in our tumors. Single-cell sequencing is allowing us to validate and narrow the scope of this hypothesis, by providing transcript expression data that can be filtered based on cell type. The ultimate goal of this project is to identify novel pro-angiogenic transcripts secreted by the small population of immune cells within the tumor, whose expression would be lost in bulk analysis.
Monday, November 6, 2017:
Monday, December 4, 2017
Presenter: Sara Linker; Title: Single-nucleus RNA-seq (snRNA-seq) reveals a unique transcriptional response in the dentate gyrus in response to hippocampal activation;
Summary: Dentate granule cells (DGCs) are sparsely active within the mouse hippocampus. Through snRNA-seq we have examined the transcriptional response of hippocampal neurons to activity and identified that, despite their rare probability of activation, DGCs have an exaggerated activity-induced transcriptional response compared to other hippocampal populations. These results highlight the importance of the baseline cell state in determining the transcriptional response to neural activity.