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Figure 5 | Genome Biology

Figure 5

From: Evidence for the biogenesis of more than 1,000 novel human microRNAs

Figure 5

Design of the custom SureSelect miRNA capture system. (a) The workflow of the capture system. An sRNA-seq library contains miRNAs of interest and other small RNAs. The library is mixed with biotinylated cRNA baits in solution. The baits are complementary in sequence to the target miRNAs and specifically hybridize with these. Magnetic streptavidin-coated beads are added to the solution and bind to the biotin. Last, the baits and their bound targets are isolated by magnetic separation, and the target miRNA clones can be sequenced. (b) The short size of miRNAs requires specific design considerations, since the SureSelect baits are much longer, at 120 nucleotides. If the baits are designed to hybridize with the target miRNA only, the interaction might be weak and targets lost. If baits are complementary to the target miRNA and the entire length of the flanking ligation adapters, which are added during library preparation, then specificity might be lost because most binding is to the universal adapter sequences. We have designed the baits so that they are complementary to the target miRNA and part of the adapters, giving a strong and specific hybridization. For targets longer than typical miRNAs, the length of the hybridized region is kept constant by dynamically varying the part of the adapters which is baited. NGS, next-generation sequencing.

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