Candidate miRNAs were identified using miRDeep2 software (47 (link)). Only miRNAs supported by >5 reads were reported in this study. AGO-CLIP data were mapped to the mouse genome using STAR (same as for small RNA-seq libraries described in Data Processing ) and the reads falling within the putative miRNA coordinates were counted using featureCounts. We counted a putative miRNA as “supported” if it had >5 AGO-CLIP counts.
To search for the previous mentions of identified miRNAs, we looked up their sequences in miRCarta (85 (link)) and used the Google search engine to query the literature. Candidate miRNAs were ranked by novoMiRank scores, which we computed as described in ref. 85 (link).
For independent validation, we performed RT-qPCR using custom Small RNA TaqMan probes (Life Technologies; catalog #4398987) designed on the star consensus sequence reported by miRDeep2. We used 0.5 ng of total RNA per tissue sample supplied with Cel-mir-39 spike-in (Qiagen; catalog #339390) to perform the reactions in a final volume of 20 μL.
We analyzed tissue and sex specificity of identified miRNAs based on transcripts supported by at least 50 sequencing reads across all samples. Statistical analysis and data visualization were performed as described above for annotated miRNAs.
To search for the previous mentions of identified miRNAs, we looked up their sequences in miRCarta (85 (link)) and used the Google search engine to query the literature. Candidate miRNAs were ranked by novoMiRank scores, which we computed as described in ref. 85 (link).
For independent validation, we performed RT-qPCR using custom Small RNA TaqMan probes (Life Technologies; catalog #4398987) designed on the star consensus sequence reported by miRDeep2. We used 0.5 ng of total RNA per tissue sample supplied with Cel-mir-39 spike-in (Qiagen; catalog #339390) to perform the reactions in a final volume of 20 μL.
We analyzed tissue and sex specificity of identified miRNAs based on transcripts supported by at least 50 sequencing reads across all samples. Statistical analysis and data visualization were performed as described above for annotated miRNAs.
