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A Magnetic Bead-Integrated Chip for the Large Scale Manufacture of Normalized esiRNAs 1 1 1 1 1 1 1 Zhao Wang , Huang Huang , Hanshuo Zhang , Changhong Sun , Yang Hao , Junyu Yang , Yu Fan , Jianzhong Jeff Xi1,2* 1 Department of Biomedical Engineering, College of Engineering, Peking University, Beijing, China, 2 State Key Laboratory of Transducer Technology, Chinese Academy of Sciences, Shanghai, China Abstract The chemically-synthesized siRNA duplex has become a powerful and widely used tool for RNAi loss-of-function studies, but suffers from a high off-target effect problem. Recently, endoribonulease-prepared siRNA (esiRNA) has been shown to be an attractive alternative due to its lower off-target effect and cost effectiveness. However, the current manufacturing method for esiRNA is complicated, mainly in regards to purification and normalization on a large-scale level. In this study, we present a magnetic bead-integrated chip that can immobilize amplification or transcription products on beads and accomplish transcription, digestion, normalization and purification in a robust and convenient manner. This chip is equipped to manufacture ready-to-use esiRNAs on a large-scale level. Silencing specificity and efficiency of these esiRNAs were validated at the transcriptional, translational and functional levels. Manufacture of several normalized esiRNAs in a single well, including those silencing PARP1 and BRCA1, was successfully achieved, and the esiRNAs were subsequently utilized to effectively investigate their synergistic effect on cell viability. A small esiRNA library targeting 68 tyrosine kinase genes was constructed for a loss-of-function study, and four genes were identified