IsoSeek for unbiased and UMI-informed sequencing of miRNAs from low input samples at single-nucleotide resolution

Abstract

Besides canonical microRNAs (miRNAs), sequence-based variations called iso-miRs have biological relevance and diagnostic potential; however, accurate calling of these post-transcriptional modifications is challenging, especially for low input samples. Here, we present IsoSeek, a sequencing protocol that reduces ligation and PCR amplification bias and improves the accuracy of miRNA detection in low input samples. We describe steps for using randomized adapters combined with unique molecular identifiers (UMI), library quantification, and sequencing, followed by detailed procedures for data processing and analysis.