Procedure for a high throughput RNAi screen for the second phase of the iBeetle screen

在这份提案中，我们详细描述了我们为iBeetle开发的筛选程序，以及两个筛选中心进行这一全基因组RNAi筛选所需的资源。如本申请的A部分概念和目标中所述，该程序已在试点屏幕中进行了测试和优化。在这项提案中，我们申请了对大约一半的Tribolium基因组进行功能分析所需的资金：在第一个赠款期间，我们打算测试6000个基因的dsRNA。为了使筛选的产量相对于投入的努力最优化，每个dsRNA将同时注射到幼虫和蛹中。通过这种方法，可以检测到胚胎发育和变态过程中许多不同过程中的基因功能。在两个中心进行筛查将通过集中实际专业知识来确保高效率，确保统一的筛查标准，并将允许学生、助教和博士后(筛查员)在分析/表型检测方面进行分工和专业化-此外，在两个中心进行筛查可以最大限度地利用现有的基础设施。

In this proposal we describe in detail the screening procedure we developed for iBeetle, and the resources required at the two screening centers to conduct this genome-wide RNAi screen. As described in Part A Concept & Aims of this application, this procedure has been tested and optimized in a pilot screen. In this proposal we apply for the funds necessary to functionally analyze about half of the Tribolium genome: In the first grant period, we intend to test dsRNAs of 6,000 genes. To optimize the yield of the screen relative to the effort invested, each dsRNA will be injected into both, larvae and pupae. By this means, gene functions in a number of different processes during embryonic development as well as metamorphosis can be detected. Screening in two centers will ensure high efficiency by concentrating the practical expertise, will ensure uniform screening criteria, and will allow for division of labor and specialization in analysis/phenotype detection among the students, the TAs and the postdoc involved (the screeners )- Moreover, screening at two centers allows maximal use of the existing infrastructure.