Journal: Nucleic Acids Research
Article Title: YAMAT-seq: an efficient method for high-throughput sequencing of mature transfer RNAs
Figure Lengend Snippet: Adapter-tRNA ligation efficiencies with a conventional method and YAMAT-seq. ( A ) Schematic representation of the adapter ligation reactions used in the conventional and YAMAT-seq methods. In the conventional method, tRNA was subjected to 3΄-adapter (3΄-AD) and 5΄-adapter (5΄-AD) ligations catalyzed by truncated Rnl2 and Rnl1, respectively, using the Illumina TruSeq Small RNA Sample Preparation Kit. In contrast, in the YAMAT-seq method, a Y-shaped adapter (Y-AD) is ligated to a tRNA by Rnl2. ( B ) Ligation products of synthetic human cyto tRNA AspGUC and 5΄-AD or 3΄-AD were quantified by real-time qRT-PCR. The amounts of ligation products were determined based on standard curves, and ligation efficiencies were calculated as percentages of ligated tRNA versus input tRNA (set as 100%). Each data set represents the average of three independent experiments with bars showing the SD. ( C ) Ligation products of the indicated native cyto tRNAs in BT-474 total RNA and 5΄-AD or 3΄-AD were quantified by real-time qRT-PCR. Each data set represents the average Ct values of three independent experiments with bars showing the SD. ( D ) Image of 8% native polyacrylamide gel electrophoresis (PAGE) of amplified cDNAs resulting from BT-474 total RNA sequencing by conventional and YAMAT-seq procedures. The total adapter lengths of the conventional and YAMAT-seq procedures are 118 and 140 bp, respectively. The YAMAT-seq-derived cDNAs in the designated Bands 1–3 regions were gel-purified and subjected to cloning; the identified sequences are shown in Supplementary Table S1 . The cDNAs in regions designated with gray lines are expected to contain tRNA fraction.
Article Snippet: For conventional ligation, 2 pmol of synthetic tRNA was subjected to 5΄- and 3΄- adapter ligation using the TruSeq Small RNA Sample Preparation Kit (Illumina).
Techniques: Ligation, Sample Prep, Quantitative RT-PCR, Polyacrylamide Gel Electrophoresis, Amplification, RNA Sequencing Assay, Derivative Assay, Purification, Clone Assay