Cell-type specific sequencing of microRNAs from complex animal tissues III

MicroRNAs (miRNAs) play an essential role in the post-transcriptional regulation of animal development and physiology. However, in vivo studies linking miRNA-function to the biology of distinct cell types within complex tissues remain challenging, partly due to the difficulties in achieving cellular resolution using available miRNA-profiling methods. Here, we report an in vivo small RNA-tagging approach that enables high-throughput sequencing of tissue- and cell type-specific miRNAs in animals, which we call microRNome by methylation-dependent sequencing, or mime-seq. The method combines orthogonal, cell-type-specific 3´ terminal 2’-O-methylation of animal miRNAs by the plant-specific methyltransferase HEN1, with oxidation-sensitive small RNA cloning and high-throughput sequencing. We show that mime-seq uncovers the miRNomes of rare cells within C. elegans and Drosophila at unprecedented specificity and sensitivity, enabling miRNA profiling with single-cell resolution in whole animals. Mime-seq overcomes the current challenges in tissue- and cell-type-specific small RNA profiling and provides novel entry points for understanding the function of miRNAs in spatially restricted physiological settings. 6 small RNA libraries from adult (2-5 days old) female Drosophila melanogaster. For all of the samples (3+3 libraries) there are two conditions (untreated and treated with sodium periodate): 2 libraries for wild-type, non-transgenic flies; 2 libraries for MHC-Gal4; UAST::AthHEN1 flies; and 2 libraries for Act88F-Gal4; UAST::AthHEN1 flies.