tRNA-seq of Blastocrithidia nonstop p57 cytosolic fraction
For tRNA isolation over 2g total RNA was resolved on urea-polyacrylamide gels and recovered within a size window of 60-100 nt for tRNA followed by coupled nucleotide demethylation and Hydro-tRNAseq method to further improve the efficiency of tRNA-seq library preparation, containing the following treatments: a) m1A and m3C demethylation of purified tRNA; b) limited alkaline hydrolysis of demethylated tRNA by carbonate buffer; c) calf intestinal phosphatase (CIP) dephosphorylation and then T4 polynucleotide kinase (T4 PNK) re-phosphorylation of the partially hydrolyzed tRNA fragments. Fragments of 19-35nt for partially hydrolyzed and re-phosphorylated tRNA were converted to small RNA sequencing libraries using NEBNext Multiplex Small RNA Library Prep Set for Illumina kit.