Identification and characterization of lepidopteran insect telomerase RNA
Spodoptera frugiperda (Sf9 cell line) or Trichoplusia ni (Hi5 cell line) telomerase was purified by a two step chromatographic strategy and telomerase activity was followed by Telomere Repeat Amplification Protocol (TRAP) through the purification steps. After the second chromatographic step, RNA co-purifying with peak activity fraction and a telomerase negative control fraction were extracted and analyzed by Illumina next-generation sequencing (NGS). Briefly Illumina libraries were constructed using the ScriptSeq v2 RNA-seq library preparation kit (Epicentre) and sequenced using the NextSeq 500 instrument. Adapters were trimmed and read pairing was maintained with trimmomatic. Processed reads were subject to a custom bioinformatics strategy to identify S. frugiperda telomerase RNA (TR) candidates and further experiments were performed to validate the bonafide S. frugiperda TR. Oxford Nanopore sequencing was applied to comprehensively map the S. frugiperda TR 3' ends. Briefly, S. frugiperda total RNA was guanosine / inosine tailed and reverse transcribed using an oligo-dC reverse primer that contains a 5' adapter. Kinased PCR primers targeting S. frugiperda TR and the oligo-dC reverse primer 5' adapter were used for PCR ampilification using a DNA polymerase that adds non-templated A residues to the 3' ends of the PCR DNA. Nanopore adapters from the SQK-LSK110 kit were ligated according to recommendations and sequenced on a Flongle flow cell. Data were basecalled using Guppy version 6.3.8 and reads that pass the quality threshold were used for 3' end mapping of S. frugiperda TR.