Genetic Engineering & Biotechnology News

AUG 2017

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Genetic Engineering & Biotechnology News | GENengnews.com | AUGUST 2017 | 19 simplified workflow for targeted, single-cell gene-expression studies. The workflow begins with dissociating a tumor or tissue sample with BD TuDoR. This reagent has been optimized to minimize cell death, delivering high yield such that the tu- mor heterogeneity is maintained. The result- ing cell suspension can either be loaded direct- ly into a BD single-cell cartridge, or can first be enriched by FACS for specific cell types (for example, tumor or T cells). The cell suspen- sion is made such that 1 out of every 10 wells of the cartridge contains a cell (approximately 20,000 cells total). Next, bead libraries are added to the car- tridge. Each bead library contains a universal PCR priming site, a cell label, a UMI, and an mRNA capture sequence of oligo dT (Figure 1). This schema ensures that each cell, and each mRNA molecule within each cell, is tagged with a unique combination of labels. Each cell is then lysed and its mRNA molecules hy- bridized to the probes on the bead. Beads are magnetically retrieved from the cartridge into a single tube. Reverse transcription and library preparation across all cells from the sample are performed in this single tube. BD single-cell gene-expression assays are designed for targeted RNA sequencing of up to 500 genes at a time. Targeted panels re- quire significantly less reads per cell vs. whole transcriptome sequencing to obtain the re- quired sequencing depth for gene-expression analysis of relevant genes. This robust tool enables the identification and characteriza- tion of novel and rare cell types within highly heterogeneous populations at a fraction of the cost of whole transcriptome sequencing. Both custom and predefined gene-expression pan- els are available through the solution. The benefits of the aforementioned assay design include the ability to sub-sample from the cDNA/bead library, and the ability to ar- chive the cDNA on the magnetic beads for at least three months (per ongoing stability stud- ies). With sub-sampling, a small portion of the cDNA/bead library can be amplified and se- quenced shallowly to determine sample qual- ity before dedicating a full sequencing run. The ability to archive cDNA/bead libraries allows users to revisit archived materials (for instance, after an initial round of analysis) for amplification with additional genes of interest. The data in Figure 2 show the tumor het- erogeneity uncovered with the BD single-cell gene-expression targeted assay. 3 In this study, cell suspensions of dissociated primary tumor and lymph node metastasis from four patients were first stained with propidium iodide and then sorted with the BD FACSAria™ II to en- rich for live cells. Next, targeted RNA libraries for each cell suspension were generated using the BD single-cell Onco-BC panel. This vali- dated, predesigned panel contains ~400 genes associated with breast cancer tumor biology. As the plots in Figure 2 show, the BD single-cell Onco-BC panel reveals numerous cell types contained within all samples, of varying proportion. Direct comparison of a set of primary and metastatic breast cancer cells revealed distinct gene-expression pat- terns in tumor cells, such as a higher propor- tion of the metastatic cells expressing cancer stem cell (CSC) gene signatures (Figure 3). Figure 4 compares the required reads per cell between a targeted panel (BD single cell Onco-BC Panel) and whole transcriptome se- quencing. Graph A shows the difference in reads sequenced per cell per assay for a breast cancer tumor sample. Graph B shows the re- sults for the UMI counts for the BD single cell Onco-BC Panel genes expressed in both the tar- geted assay and whole transcriptome libraries, normalized to sequencing reads. The total sum of molecules associated with the genes in the BD single cell Onco-BC Panel accounts for only 4.6% of molecules observed in the whole tran- scriptome data. These data show that targeted assay libraries require as little as ~2% of the sequencing reads as whole transcriptome librar- ies to achieve the same sequencing depth for cell-type identification and analysis of selected genes of interest, presenting an economical al- ternative to whole transcriptome sequencing. The aforementioned data show the ben- efits of the BD single-cell analysis system, to- gether with BD TuDoR and BD FACS. This solution provides the complete tools for ef- fective, efficient, and economical single-cell analysis of tumor heterogeneity and for the elucidation of disease mechanism. Disclaimer: BD single-cell gene-expression platform, BD TuDoR, and BDFACSAria are for research use only and not intended for use in diagnostic or therapeutic procedures. References available online. Discovery on Target .com TARGET Disc very on The Industry's Preeminent Event on Novel Drug Targets 15 th Annual September 25-29, 2017 • The Westin Copley Place • Boston, MA Cancer Immunotherapy » Immunomodulatory Small Molecules » Microbiome in Immuno-Oncology » NK Cell-Based Cancer Immunotherapy » Targeting Tumor Myeloid Cells Target-Based Discovery & Validation » Targeting Histone Methyltransferases & Demethylases » Targeting the Ubiquitin Proteasome System » Lead Generation Strategies » CRISPR for Disease Modeling & Target Discovery » GPCR-Based Drug Discovery » Next-Generation Histone Deacetylase Inhibitors » Kinase Inhibitor Discovery » Target Identification Strategies Hot & Emerging » Targeting Autophagy » Targeting HBV » Targeting the Microbiome » NASH & Fibrosis » Autoimmune & Inflammation Drug Targets » Targeting Ocular Disorders » CNS & Neurodegenerative Targets » Tackling Rare Diseases Biologics & Beyond » Constrained Peptides & Macrocyclics » Antibodies Against Membrane Protein Targets - Part 1 » Antibodies Against Membrane Protein Targets - Part 2 » Emerging Oligonucleotide Therapeutics Mention keycode GEN100 when registering SAVE $100 OFF CURRENT RATE Kathleen Shelton, is director of market development; Beth Walczak, Ph.D., (ewalczak@Cellular-Research.com) is R&D applications manager, BD Genomics, Menlo Park, CA. Website: www.bd.com OMICS Assay Tutorial

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