GSE104466
Cell-type specific sequencing of microRNAs from complex animal tissues II
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. Overall design: two replicates with 12 libraries each: 12 libraries of 10-fold decreasing dilution series of RNA from S2-S cells (DmHen1-KO) expressing AthHEN1 into RNA from Mmu ES cells:  2 libraries with RNA from S2-S cells (DmHen1-KO) expressing AthHEN1 mixed in 1:1 ratio with RNA from Mmu ES cells, one subjected to periodate treatment (ox+) and one left untreated. 2 libraries with RNA from S2-S cells (DmHen1-KO) expressing AthHEN1 mixed in 1:10 ratio with RNA from Mmu ES cells, one subjected to periodate treatment (ox+) and one left untreated. 2 libraries with RNA from S2-S cells (DmHen1-KO) expressing AthHEN1 mixed in 1:100 ratio with RNA from Mmu ES cells, one subjected to periodate treatment (ox+) and one left untreated. 2 libraries with RNA from S2-S cells (DmHen1-KO) expressing AthHEN1 mixed in 1:1000 ratio with RNA from Mmu ES cells, one subjected to periodate treatment (ox+) and one left untreated. 2 libraries with RNA from S2-S cells (DmHen1-KO) expressing AthHEN1 mixed in 1:10,000 ratio with RNA from Mmu ES cells, one subjected to periodate treatment (ox+) and one left untreated. 2 libraries with RNA from S2-S cells (DmHen1-KO) expressing AthHEN1 mixed in 1:100,000 ratio with RNA from Mmu ES cells, one subjected to periodate treatment (ox+) and one left untreated.