miRNA Induced Silencing Complex Immunoprecipitation, RNA Sequencing Library Preparation, Sequencing, and Analysis

miRNA induced silencing complex (miRISC) immunoprecipitation was performed as previously described (8) with a mouse–anti–AGO2 antibody (clone E12–1C9; Abnova). Library preparation from mRNA enriched from total RNA with a NEBNext Poly(A) mRNA Magnetic Isolation Kit (New England Biolabs) and from miRISC-isolated RNA was performed using a NEBNext Ultra II Directional RNA Library Prep Kit from Illumina. Library preparation from human islets total RNA (200 ng) was performed using a NEBNext Low Input RNA Library Prep kit (New England Biolabs). Libraries were sequenced on a HiSeq4000. Mapping and differential expression analysis were performed with Salmon v1.3.0 (9) and DESeq2 (10).

For miRNA target identification, total mRNA (T-RNA) and immunoprecipitated RNA (RIP-RNA) samples were treated as separate data sets, and the two resulting gene lists of differential analysis were used to calculate the ratio of RIP-RNA to T-RNA for each gene. Additional details are provided in Supplementary Research Design and Methods.