{"id":1804,"date":"2024-04-04T09:50:02","date_gmt":"2024-04-04T09:50:02","guid":{"rendered":"https:\/\/skyangbio.com\/wordpress\/?page_id=1804"},"modified":"2024-04-14T05:25:22","modified_gmt":"2024-04-14T05:25:22","slug":"pooled-shrna-library","status":"publish","type":"page","link":"https:\/\/skyangbio.com\/wordpress\/pooled-shrna-library\/","title":{"rendered":"Pooled sHRNA Library"},"content":{"rendered":"\t\t<div data-elementor-type=\"wp-page\" data-elementor-id=\"1804\" class=\"elementor elementor-1804\" data-elementor-post-type=\"page\">\n\t\t\t\t<div class=\"elementor-element elementor-element-6c92bdf e-flex e-con-boxed e-con e-parent\" data-id=\"6c92bdf\" data-element_type=\"container\">\n\t\t\t\t\t<div class=\"e-con-inner\">\n\t\t<div class=\"elementor-element elementor-element-c70f853 e-con-full e-flex e-con e-child\" data-id=\"c70f853\" data-element_type=\"container\">\n\t\t\t\t<\/div>\n\t\t<div class=\"elementor-element elementor-element-8f33224 e-con-full e-flex e-con e-child\" data-id=\"8f33224\" data-element_type=\"container\">\n\t\t\t\t<div class=\"elementor-element elementor-element-d00662e elementor-widget elementor-widget-heading\" data-id=\"d00662e\" data-element_type=\"widget\" data-widget_type=\"heading.default\">\n\t\t\t\t<div class=\"elementor-widget-container\">\n\t\t\t\t\t<h2 class=\"elementor-heading-title elementor-size-default\">Pooled shRNA libraries<\/h2>\t\t\t\t<\/div>\n\t\t\t\t<\/div>\n\t\t\t\t<\/div>\n\t\t\t\t\t<\/div>\n\t\t\t\t<\/div>\n\t\t<div class=\"elementor-element elementor-element-c2c6cb8 e-flex e-con-boxed e-con e-parent\" data-id=\"c2c6cb8\" data-element_type=\"container\">\n\t\t\t\t\t<div class=\"e-con-inner\">\n\t\t\t\t<div class=\"elementor-element elementor-element-6804dc7 elementor-tabs-view-horizontal elementor-widget elementor-widget-tabs\" data-id=\"6804dc7\" data-element_type=\"widget\" data-widget_type=\"tabs.default\">\n\t\t\t\t<div class=\"elementor-widget-container\">\n\t\t\t\t\t\t\t<div class=\"elementor-tabs\">\n\t\t\t<div class=\"elementor-tabs-wrapper\" role=\"tablist\" >\n\t\t\t\t\t\t\t\t\t<div id=\"elementor-tab-title-1091\" class=\"elementor-tab-title elementor-tab-desktop-title\" aria-selected=\"true\" data-tab=\"1\" role=\"tab\" tabindex=\"0\" aria-controls=\"elementor-tab-content-1091\" aria-expanded=\"false\">Pooled screening overview<\/div>\n\t\t\t\t\t\t\t\t\t<div id=\"elementor-tab-title-1092\" class=\"elementor-tab-title elementor-tab-desktop-title\" aria-selected=\"false\" data-tab=\"2\" role=\"tab\" tabindex=\"-1\" aria-controls=\"elementor-tab-content-1092\" aria-expanded=\"false\">Overview<\/div>\n\t\t\t\t\t\t\t\t\t<div id=\"elementor-tab-title-1093\" class=\"elementor-tab-title elementor-tab-desktop-title\" aria-selected=\"false\" data-tab=\"3\" role=\"tab\" tabindex=\"-1\" aria-controls=\"elementor-tab-content-1093\" aria-expanded=\"false\">Pooled screening deconvolution<\/div>\n\t\t\t\t\t\t\t<\/div>\n\t\t\t<div class=\"elementor-tabs-content-wrapper\" role=\"tablist\" aria-orientation=\"vertical\">\n\t\t\t\t\t\t\t\t\t<div class=\"elementor-tab-title elementor-tab-mobile-title\" aria-selected=\"true\" data-tab=\"1\" role=\"tab\" tabindex=\"0\" aria-controls=\"elementor-tab-content-1091\" aria-expanded=\"false\">Pooled screening overview<\/div>\n\t\t\t\t\t<div id=\"elementor-tab-content-1091\" class=\"elementor-tab-content elementor-clearfix\" data-tab=\"1\" role=\"tabpanel\" aria-labelledby=\"elementor-tab-title-1091\" tabindex=\"0\" hidden=\"false\"><h2 class=\"yikes-custom-woo-tab-title yikes-custom-woo-tab-title-pooled-screening-overview\">Pooled screening overview<\/h2><p>In pooled shRNA screening, hundreds to thousands of hairpins are combined (pooled) and interrogated simultaneously in a multiplex assay without the need for robotics or liquid handling.<br \/>The schematic to the below depicts this process.<\/p><ul><li>1. Transduction:\u00a0Cells are transduced at a low MOI ensuring a single shRNA is expressed in each cell.<\/li><li>2. Screen:\u00a0The screen employs a selection process that is specific to the researcher\u2019s assay.\u00a0 There are two underlying strategies for selection:\u00a0Negative selection (dropout) screens\u00a0include an untreated control for comparison to allow the detection of shRNA that provided resistance or sensitized the cells to the selective reagent.\u00a0Positive selection (enrichment) screens\u00a0only detect surviving cells and do not require an untreated control.<\/li><li>3. Analysis:\u00a0Under selection, resistant cells increase in the population and sensitized cells decrease in the population.\u00a0 These changes in representation can be detected by sequence analysis (either Sanger sequencing or Next Generation Sequencing).<\/li><\/ul><p><img fetchpriority=\"high\" decoding=\"async\" class=\"alignnone wp-image-2288 size-full\" src=\"https:\/\/skyangbio.com\/wordpress\/wp-content\/uploads\/2024\/04\/Pooled-Screening-library.png\" alt=\"\" width=\"500\" height=\"455\" srcset=\"https:\/\/skyangbio.com\/wordpress\/wp-content\/uploads\/2024\/04\/Pooled-Screening-library.png 500w, https:\/\/skyangbio.com\/wordpress\/wp-content\/uploads\/2024\/04\/Pooled-Screening-library-200x182.png 200w, https:\/\/skyangbio.com\/wordpress\/wp-content\/uploads\/2024\/04\/Pooled-Screening-library-300x273.png 300w\" sizes=\"(max-width: 500px) 100vw, 500px\" \/><\/p><h2>Pooled screening assays<\/h2><p>The underlying workflow of all pooled shRNA screens begins by transducing cells with a heterogeneous pool of viral particles followed by an assay that enriches specific cells based on a phenotypic change.\u00a0 Many variations have been successfully used\u00a0in vivoor\u00a0in vitro. However, most fall into three categories:\u00a0 viability screen, reporter screen or behavioral screen1,2<\/p><h5><span style=\"font-weight: bold;\">Viability Screen:<\/span><\/h5><p>In viability screens, the pool is most often used in combination\u00a0 with a selective agent (e.g. drug treatment, exposure to pathogens).\u00a0 An shRNA\u2019s impact on cell health is measured by changes in its representation in population.\u00a0 A negative impact on cell viability will decrease an shRNA\u2019s representation while a positive impact on cell viability will increase representation3,4,5.<\/p><h5><span style=\"font-weight: bold;\">Reporter Screens:<\/span><\/h5><p>Reporters, such as fluorescent markers, can be used to indicate changes in transcription or can be fused to proteins to measure stability or localization.\u00a0 Cells can be sorted for high or low levels of expression and hairpins from each group can be analyzed for their enrichment or depletion8.<\/p><h5><span style=\"font-weight: bold;\">Behavioral Screens:<\/span><\/h5><p>Cell behavior can also be assessed in a pooled screening assay.\u00a0 For example, colony formation on soft agar can be used to select for cells with anchorage independent growth which can be an indicator of oncogenic transformation9,10.<\/p><p>In addition, one of the advantages of shRNA over siRNA is the ability to perform these screens\u00a0in vivo\u00a0as well as\u00a0in vitro.\u00a0 Viability screens have been successfully adapted using xenographs to identify tumor suppressor genes as well as oncogenes5,6,7.<\/p><p>This range of application in addition to the accessibility of shRNA pools provides researchers with a powerful option for screening.<\/p><h2 class=\"yikes-custom-woo-tab-title yikes-custom-woo-tab-title-documents\">Documents<\/h2><h5>Technical manual<\/h5><p style=\"clear: both;\"><a href=\"http:\/\/www.moldiag.in\/wp-content\/uploads\/2016\/06\/Lentiviral-shRNA-Pooled-Screening-Library-Plasmid-Format-Tech-Manual.pdf\" target=\"_blank\" rel=\"noopener\"><span style=\"font-weight: bold;\">shERWOOD-Ultramir Lentiviral shRNA Pooled Library \u2013 Plasmid<\/span><\/a><br \/><a href=\"http:\/\/www.moldiag.in\/wp-content\/uploads\/2016\/06\/Lentiviral-shRNA-Pooled-Screening-Library-Viral-Particle-Format-Tech-Manual.pdf\" target=\"_blank\" rel=\"noopener\"><span style=\"font-weight: bold;\">shERWOOD-Ultramir Lentiviral shRNA Pooled Library \u2013 Viral Particles<\/span><\/a><br \/><a href=\"http:\/\/www.moldiag.in\/wp-content\/uploads\/2016\/06\/Promoter-selectioin-kit-pZIP-Lenitviral-Vectors-Technical-Manual.pdf\" target=\"_blank\" rel=\"noopener\"><span style=\"font-weight: bold;\">ZIP Promoter Selection Kit<\/span><\/a><\/p><h5><span style=\"font-weight: bold;\">References<\/span><\/h5><p><a href=\"http:\/\/www.ncbi.nlm.nih.gov\/pmc\/articles\/PMC3333774\/\" target=\"_blank\" rel=\"noopener\"><span style=\"font-weight: bold;\">1. \u00a0Sims D. et al.\u00a0 (2011) High-throughput RNA interference screening using pooled shRNA libraries and next generation sequencing. Genome Biology, 12(10):R104<\/span><\/a><br \/><a href=\"http:\/\/www.ncbi.nlm.nih.gov\/pubmed\/22271906\" target=\"_blank\" rel=\"noopener\"><span style=\"font-weight: bold;\">2. Hu G and Luo J. (2012) A primer on using pooled shRNA libraries for functional genomic screens. \u00a0Acta Biochim Biophys Sin. Feb;44(2):103-12<\/span><\/a><br \/><a href=\"http:\/\/www.ncbi.nlm.nih.gov\/pmc\/articles\/PMC3244037\/\" target=\"_blank\" rel=\"noopener\"><span style=\"font-weight: bold;\">3. Westerman B. et al. (2011) A genome-wide RNAi screen in mouse embryonic stem cells identifies Mp1 as a key mediator of differentiation. J Exp Med. Dec 19; 208(13): 2675\u20132689<\/span><\/a><br \/><a href=\"http:\/\/www.sciencemag.org\/content\/319\/5863\/620.figures-only\" target=\"_blank\" rel=\"noopener\"><span style=\"font-weight: bold;\">4. Schlabach M. et al. (2008) Cancer Proliferation Gene Discovery Through Functional Genomics Science 1 February 2008: Vol. 319 no. 5863 pp. 620-624<\/span><\/a><br \/><a href=\"http:\/\/www.cell.com\/cell\/abstract\/S0092-8674(09)00529-7\" target=\"_blank\" rel=\"noopener\"><span style=\"font-weight: bold;\">5. Luo, J. et al.\u00a0 (2009) A Genome-wide RNAi Screen Identifies Multiple Synthetic Lethal Interactions with the Ras Oncogene. Cell Volume 137, Issue 5, p835\u2013848, 29 May<\/span><\/a><br \/><a href=\"http:\/\/www.ncbi.nlm.nih.gov\/pubmed\/?term=Wuestefeld%2C+T.+et+al.+A+Direct+In+Vivo+RNAi+Screen+Identifies+MKK4+as+a+Key+Regulator+of+Liver+Regeneration.++Volume+153%2C+Issue+2%2C+11+April+2013%2C+Pages+389%E2%80%93401\" target=\"_blank\" rel=\"noopener\"><span style=\"font-weight: bold;\">6. Wuestefeld, T. et al. (2013) A Direct In Vivo RNAi Screen Identifies MKK4 as a Key Regulator of Liver Regeneration.\u00a0 Cell Apr 11; 153(2): 389\u2013401<\/span><\/a><br \/><a href=\"http:\/\/www.ncbi.nlm.nih.gov\/pubmed\/21760589\" target=\"_blank\" rel=\"noopener\"><span style=\"font-weight: bold;\">7. Possemato, R. et al. (2011) Functional genomics reveal that the serine synthesis pathway is essential in breast cancer. Nature Aug 18; 476(7360): 346\u2013350<\/span><\/a><br \/><a href=\"http:\/\/www.ncbi.nlm.nih.gov\/pubmed\/?term=Zender%2C+L.+et+al.+An+Oncogenomics-Based+In+Vivo+RNAi+Screen+Identifies+Tumor+Suppressors+in+Liver+Cancer.+Cell.+Volume+135%2C+Issue+5%2C+28+November+2008%2C+Pages+852%E2%80%93864\" target=\"_blank\" rel=\"noopener\"><span style=\"font-weight: bold;\">8. Zender, L. et al. (2008) An Oncogenomics-based in vivo RNAi screen identifies tumor suppressors in liver cancer. Cell. Nov 28; 135(5): 852\u2013864<\/span><\/a><br \/><a href=\"http:\/\/www.ncbi.nlm.nih.gov\/pmc\/articles\/PMC2147719\/\" target=\"_blank\" rel=\"noopener\"><span style=\"font-weight: bold;\">9. Gazin , C. et al. (2007) An Elaborate Pathway Required for Ras-Mediated Epigenetic Silencing. Nature, Oct 25; 449(7165): 1073-1077<\/span><\/a><br \/><a href=\"http:\/\/genesdev.cshlp.org\/content\/24\/23\/2654.full.pdf\" target=\"_blank\" rel=\"noopener\"><span style=\"font-weight: bold;\">10. Smolen G. et al. (2010) A genome-wide RNAi screen identifies multiple RSK-dependent regulators of cell migration. Genes Dev ;24:v2654-2665.<\/span><\/a><br \/><a href=\"http:\/\/www.ncbi.nlm.nih.gov\/pubmed?term=Westbrook%20T%20%20et%20al.%20A%20genetic%20screen%20for%20candidate%20tumor%20suppressors%20identifies%20REST.%20Cell.%202005;121:837%E2%80%93848.%5ball%5d&amp;cmd=correctspelling\" target=\"_blank\" rel=\"noopener\"><span style=\"font-weight: bold;\">11. Westbrook T. et al. (2005) A genetic screen for candidate tumor suppressors identifies REST. Cell. 121(6): 837\u2013848.<\/span><\/a><\/p><\/div>\n\t\t\t\t\t\t\t\t\t<div class=\"elementor-tab-title elementor-tab-mobile-title\" aria-selected=\"false\" data-tab=\"2\" role=\"tab\" tabindex=\"-1\" aria-controls=\"elementor-tab-content-1092\" aria-expanded=\"false\">Overview<\/div>\n\t\t\t\t\t<div id=\"elementor-tab-content-1092\" class=\"elementor-tab-content elementor-clearfix\" data-tab=\"2\" role=\"tabpanel\" aria-labelledby=\"elementor-tab-title-1092\" tabindex=\"0\" hidden=\"hidden\"><h2>Choice of pomoter,reporter, gene content and more<\/h2><h5><strong>Achieve superior RNAi screens with enhanced potency, sequence-verified \u00a0shRNA pools and flexible delivery strategies.\u00a0<\/strong><\/h5><table><tbody><tr><td>Greater confidence in your results<\/td><td><ul><li>Every clone is sequence verified \u2013 eliminate unwanted background from mutations introduced in chip-based pools<\/li><li>More potent shRNA per gene \u2013 decrease false positive and false negative results<\/li><li>Equimolar pooling process \u2013 reduces variation between samples<\/li><\/ul><\/td><\/tr><tr><td>Choose the best promoter for your cells<\/td><td><ul><li>Select the optimal promoter for your target cell line with a choice of promoters- Choose from SFFV,\u00a0hCMV, mCMV, hEF1-alpha, mEF1-alpha and more<\/li><\/ul><\/td><\/tr><tr><td>Your choice of format<\/td><td><ul><li>In vitro\u00a0or\u00a0in vivo\u00a0pool formats.\u00a0 Pools can be subdivided into minipools for lower complexity and higher representation for\u00a0in vivo\u00a0or other applications that require\u00a0less complex pools<\/li><li>Choice of gene coverage \u2013 premade gene families or custom gene sets<\/li><li>Ready to use lentiviral particles\u00a0or plasmid DNA format<\/li><\/ul><\/td><\/tr><tr><td>Pool Deconvolution Service<\/td><td><ul><li>NGS analysis service \u2013 send us your genomic DNA and receive counts for every shRNA construct<\/li><li>Optimized\u00a0indexed primer kit available for NGS\u00a0(Illumina platform)<\/li><\/ul><\/td><\/tr><\/tbody><\/table><h2>Flexible pool formats for optimal pooled shRNA screens\u00a0<\/h2><p>In vitro, in vivo\u00a0and\u00a0ex vivo\u00a0screening strategies have different requirements for pool formatting.\u00a0\u00a0In vivo\u00a0and\u00a0ex vivo\u00a0screens require smaller, less complex pools to facilitate a higher shRNA representation while\u00a0in vitro\u00a0screens can be efficiently performed with higher complexity pools.\u00a0 Pooled shRNA screening libraries are available as viral particles that are ready-to-screen or plasmid DNA that can be packaged as needed for small or large-scale screens. In addition pools have been subdivided into mini pools of ~1000 shRNA per pool to enable low complexity and higher representation for\u00a0in vivo\u00a0and other screening applications.\u00a0<\/p><h5><strong>Screening capacity:<\/strong><\/h5><ul><li>Lentiviral pooled shRNA libraries provided as viral particles include enough virus to perform a screen at 500-1000-fold representation with at least 2 biological replicates.<\/li><li>Libraries provided as plasmid DNA include enough plasmid for more than 3 packaging reactions.\u00a0 Total viral particles generated will depend on packaging efficiency, but is expected to provide enough viral particles to perform a screen in multiple cell lines at 500-1000-fold representation with 3 biological replicates.<\/li><\/ul><h2>Custom pools\u00a0<\/h2><h5><strong>Custom Pooled shRNA Libraries<\/strong><\/h5><p>Custom pools can be created from any shRNA content, at titers up to 10^9TU\/ml, formatted for\u00a0in vitro,\u00a0or in vivo\u00a0screening, with a choice of promoters or fluorescent reporters.\u00a0<br \/>Contact us at\u00a0<strong><a href=\"mailto:info@skyangbio.com\">info@skyangbio.com<\/a><\/strong>\u00a0for more information and pricing<\/p><\/div>\n\t\t\t\t\t\t\t\t\t<div class=\"elementor-tab-title elementor-tab-mobile-title\" aria-selected=\"false\" data-tab=\"3\" role=\"tab\" tabindex=\"-1\" aria-controls=\"elementor-tab-content-1093\" aria-expanded=\"false\">Pooled screening deconvolution<\/div>\n\t\t\t\t\t<div id=\"elementor-tab-content-1093\" class=\"elementor-tab-content elementor-clearfix\" data-tab=\"3\" role=\"tabpanel\" aria-labelledby=\"elementor-tab-title-1093\" tabindex=\"0\" hidden=\"hidden\"><h2 class=\"yikes-custom-woo-tab-title yikes-custom-woo-tab-title-pooled-screening-deconvolution\">Pooled screening deconvolution<\/h2><h5>Pooled shRNA sequencing and deconvolution service\u00a0<\/h5><p><img decoding=\"async\" class=\"alignnone wp-image-2289 size-large\" src=\"https:\/\/skyangbio.com\/wordpress\/wp-content\/uploads\/2024\/04\/shRNA-Sequencing-min-1024x143.png\" alt=\"\" width=\"1024\" height=\"143\" srcset=\"https:\/\/skyangbio.com\/wordpress\/wp-content\/uploads\/2024\/04\/shRNA-Sequencing-min-1024x143.png 1024w, https:\/\/skyangbio.com\/wordpress\/wp-content\/uploads\/2024\/04\/shRNA-Sequencing-min-200x28.png 200w, https:\/\/skyangbio.com\/wordpress\/wp-content\/uploads\/2024\/04\/shRNA-Sequencing-min-300x42.png 300w, https:\/\/skyangbio.com\/wordpress\/wp-content\/uploads\/2024\/04\/shRNA-Sequencing-min-768x108.png 768w, https:\/\/skyangbio.com\/wordpress\/wp-content\/uploads\/2024\/04\/shRNA-Sequencing-min.png 1135w\" sizes=\"(max-width: 1024px) 100vw, 1024px\" \/><\/p><p>Need help deconvoluting your pooled screen? Send us your genomic DNA samples for sequencing and analysis.<\/p><p>\u2022\u00a0<strong>Simplify your screen<\/strong>\u00a0\u2013 Sequencing service including library preparation, next generation sequencing and data analysis<br \/>\u2022<strong>\u00a0Cost effective<\/strong>\u00a0\u2013 Multiplex sequencing allows analysis of multiple samples in parallel<br \/>\u2022\u00a0<strong>Quick turnaround<\/strong>\u00a0\u2013 4-6 weeks from receipt of samples to delivery of data<br \/>\u2022\u00a0<strong>More data reproducibility<\/strong>\u00a0\u2013 Optimized protocols and reagents for shRNA specific sequencing<\/p><p>In addition to providing the highest quality sensor-based shRNA pooled library, we also offer next generation sequencing analysis and deconvolution services. Provide us with the genomic DNA from the samples following your screen and we will provide the precise number of sequencing reads of each individual clone within a pooled shRNA sample.<\/p><p>Pricing is based on the number of samples and the number of shRNAs in a pool.<br \/>Please contact us on\u00a0<strong><a href=\"mailto:info@skyangbio.com\">info@skyangbio.com <\/a><\/strong>for further information and pricing.<\/p><\/div>\n\t\t\t\t\t\t\t<\/div>\n\t\t<\/div>\n\t\t\t\t\t\t<\/div>\n\t\t\t\t<\/div>\n\t\t\t\t\t<\/div>\n\t\t\t\t<\/div>\n\t\t<div class=\"elementor-element elementor-element-1ac236b e-flex e-con-boxed e-con e-parent\" data-id=\"1ac236b\" data-element_type=\"container\">\n\t\t\t\t\t<div class=\"e-con-inner\">\n\t\t\t\t<div class=\"elementor-element elementor-element-171bd62 elementor-widget elementor-widget-text-editor\" data-id=\"171bd62\" data-element_type=\"widget\" data-widget_type=\"text-editor.default\">\n\t\t\t\t<div class=\"elementor-widget-container\">\n\t\t\t\t\t\t\t\t\t<h2 class=\"yikes-custom-woo-tab-title yikes-custom-woo-tab-title-purchase\">Purchase<\/h2><p>Pooled shRNA libraries are available in both formats with choice of promoter and selection marker \u2013 plasmid as well as ready lentiviral particles. 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