Under-representation of 2′-O-methylated small RNAs in cDNA pools. (A) Schematic representation of ligated small RNA qPCR. Size-fractionated small RNA from mouse testis was ligated to a pre-adenylated DNA adapter. The RT primer contained a 5′-overhang region that provides a PCR priming site. QPCR was performed for specific small RNA targets in the pools using unique forward primers with a common reverse primer. (B) Relative quantification of ligated small RNA. Normalized amounts of small RNAs in cDNA pools were compared in cDNA pools prepared using T4 Rnl1 and T4 Rnl2tr. Signals corresponding to small RNAs in T4 Rnl2tr pools are shown as means ± SEM of three or more experimental replicates relative to signal from T4 Rnl1 cDNA pools. (C) Schematic representation of cloning workflow. Equimolar mixtures of unmodified or 2′-O-methylated 21-nt RNAs were ligated to a pre-adenylated DNA adapter. Input RNAs were distinguishable by reversal of the nucleotides at positions 10 and 11 (bold). Samples were further processed in parallel and subjected to sequence analysis. (D) Quantification of sequenced small RNAs. Identities of recovered clones corresponding to input small RNAs were scored in a blinded fashion. Proportions of cDNAs arising from unmodified or 2′-O-methylated RNA sequenced were compared to an expected recovery ratio of 1:1 using the exact binomial test. P-values from these tests are shown below (n = 3 experimental replicates, 248 ligation junctions sequenced).
