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

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. 46 small RNA libraries from L1-staged C. elegans. For most of the samples (40/46 libraries) there are two conditions (untreated and treated with sodium periodate) and two biological replicates: 4 libraries for wild-type, non-transgenic worms; 4 libraries for rab-3:Ath-HEN1, henn-1(0) worms; 4 libraries for unc-31:Ath-HEN1, henn-1(0) worms; 4 libraries for rgef-1:Ath-HEN1, henn-1(0) worms; 4 libraries for elt-2:Ath-HEN1, henn-1(0) worms; 4 libraries for myo-2:Ath-HEN1, henn-1(0) worms; 4 libraries for ASE:Ath-HEN1, henn-1(0) worms; 4 libraries for ASE:Ath-HEN1 worms; 4 libraries for myo-3:Ath-HEN1, henn-1(0) worms; 4 libraries for myo-3:Ath-HEN1 worms. For the remaining samples (6/46 libraries) there are two conditions (untreated and treated with sodium periodate) and three biological replicates: 6 libraries for rps-5-3:Ath-HEN1, henn-1(0) worms.