Sequencing cDNA generated from N21 RNA libraries. a Number of reads for the 100 most abundant sequences in the N21 libraries, prepared with Illumina (red) or HD adapters (blue). b-d Frequencies of predicted nucleotide base-pairing per position for N21 insert (b), N21 insert and 3’ adapter (c) and 5’ adapter, insert and 3’ adapter (d). In (c) and (d) vertical dotted line indicates ligation point. Red line denotes data obtained with Illumina protocol, blue line with HD protocol and grey line randomly generated sets of 21nt sequences. Bars indicate minimum and maximum values in all replicates. Horizontal bars at bottom indicate sequence region: green, insert; red, 3’ adapter; blue, 5’ adapter. For insert folding frequencies obtained with random sequences are more closely matched by HD data (R2 = 0.83) than by Illumina data (R2 = 0.60). e Comparison of T4 Rnl2 ligase activity on substrates with ss flaps of differing nucleotide lengths upstream or downstream of ligation site. In vitro ligation assay of RNA-DNA duplexes with either a nick (0NT) or ss flaps up- or downstream from the ligation site was carried out at 25°C for 30 min. Substrates with ss flaps >2nt in length upstream from the ligation site are inefficiently ligated. The diagram illustrates the position of the flaps, the fluorescein reporter group (star) and the backbone oligonucleotide (black). If ligation occurs the size of the nucleic acid attached to the fluorescein increases as visualised by 15% PAGE.