The adapter sequences were trimmed and reads aligned to mouse microRNAs in the miRBase database (Release 18.0)
20 using Genomics Workbench V4.0 software (CLCbio, Aarhus, Denmark), allowing two mismatches. The number of reads for each microRNA were normalized to reads per million mapped (RPM). The reads that did not match any annotated mouse microRNAs were aligned with other mammalian microRNAs to identify potential novel orthologs. To confirm that matching sequences represented novel orthologs, their genomic location and secondary structure were investigated using the UCSC genome browser (
http://genome.ucsc.edu) and RNA fold WebServer (
http://rna.tbi.univie.ac.at/cgi-bin/RNAfold.cgi). Ensemble noncoding RNA annotations, including small nucleolar RNAs (snoRNAs), for the mouse genome were downloaded using Biomart (
www.biomart.org). For the identification of putative novel microRNAs, the unannotated unique sequences were converted into FASTA format using “FASTA manipulation” in the Galaxy Web-based platform (
https://main.g2.bx.psu.edu) and submitted to mirTools Web server (
http://centre.bioinformatics.zj.cn/mirtools/).
28 The genomic location and potential secondary structure of putative novel microRNA sequences were assessed as for novel orthologs above. Publicly available small RNA sequencing data from a range of mouse tissues were accessed via the Gene Expression Omnibus database (GEO),
29 to analyze the expression level of novel microRNAs in other mouse tissues. Reads were mapped to mirtron sequences downloaded from the Eric Lai lab (
http://ericlailab.com/mammalian_mirtrons/mm9/).
30 The predicted targets and involvement in signaling pathways of the highly expressed retina- and RPE/choroid-enriched microRNAs were analyzed using DIANA miRPath V2.0,
31 and the predicted targets for isomiRs analyzed using DIANA microT V3.0.
32