Accurate characterization of Escherichia coli tRNA modifications with a simple method of deep-sequencing library preparation.
RNA Biol
; 18(1): 33-46, 2021 01.
Article
en En
| MEDLINE
| ID: mdl-32618488
ABSTRACT
In conventional RNA high-throughput sequencing, modified bases prevent a large fraction of tRNA transcripts to be converted into cDNA libraries. Recent proposals aiming at resolving this issue take advantage of the interference of base modifications with RT enzymes to detect and identify them by establishing signals from aborted cDNA transcripts. Because some modifications, such as methyl groups, do almost not allow RT bypassing, demethylation and highly processive RT enzymes have been used to overcome these obstacles. Working with Escherichia coli as a model system, we show that with a conventional (albeit still engineered) RT enzyme and key optimizations in library preparation, all RT-impairing modifications can be highlighted along the entire tRNA length without demethylation procedure. This is achieved by combining deep-sequencing samples, which allows to establish aborted transcription signal of higher accuracy and reproducibility, with the potential for differentiating tiny differences in the state of modification of all cellular tRNAs. In addition, our protocol provides estimates of the relative tRNA abundance.
Palabras clave
Texto completo:
1
Colección:
01-internacional
Base de datos:
MEDLINE
Contexto en salud:
3_ND
Problema de salud:
3_neglected_diseases
/
3_zoonosis
Asunto principal:
ARN Bacteriano
/
ARN de Transferencia
/
Regulación Bacteriana de la Expresión Génica
/
Procesamiento Postranscripcional del ARN
/
Escherichia coli
Idioma:
En
Revista:
RNA Biol
Asunto de la revista:
BIOLOGIA MOLECULAR
Año:
2021
Tipo del documento:
Article
País de afiliación:
Francia