title: tRNA sequencing of T. cruzi
design: 250 ng of purified tRNA was deacetylated in 500 L of 100 mM Tris-HCl (pH 9.0) and incubated at 37C for 1 hour. cDNA was synthesized using 5 pmol of TGIRT-III (InGex). The single-stranded cDNA was circularized with 50 units of CircLigase II, followed by an incubation at 60C for 1 hour. An additional 50 units of CircLigase II were then added, and the mixture was incubated for another hour at 60C. Finally, the cDNA was amplified using Phusion DNA polymerase (New England Biolabs) with the o231 primer and index primers.