Optimization of library preparation based on SMART for ultralow RNA-seq in mice brain tissues.
BMC Genomics
; 22(1): 809, 2021 Nov 10.
Article
en En
| MEDLINE
| ID: mdl-34758728
ABSTRACT
BACKGROUND:
Single-cell RNA sequencing (scRNA-seq) provides new insights to address biological and medical questions, and it will benefit more from the ultralow input RNA or subcellular sequencing.RESULTS:
Here, we present a highly sensitive library construction protocol for ultralow input RNA sequencing (ulRNA-seq). We systematically evaluate experimental conditions of this protocol, such as reverse transcriptase, template-switching oligos (TSO), and template RNA structure. It was found that Maxima H Minus reverse transcriptase and rN modified TSO, as well as all RNA templates capped with m7G improved the sequencing sensitivity and low abundance gene detection ability. RNA-seq libraries were successfully prepared from total RNA samples as low as 0.5 pg, and more than 2000 genes have been identified.CONCLUSIONS:
The ability of low abundance gene detection and sensitivity were largely enhanced with this optimized protocol. It was also confirmed in single-cell sequencing, that more genes and cell markers were identified compared to conventional sequencing method. We expect that ulRNA-seq will sequence and transcriptome characterization for the subcellular of disease tissue, to find the corresponding treatment plan.Palabras clave
Texto completo:
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Banco de datos:
MEDLINE
Asunto principal:
Secuenciación de Nucleótidos de Alto Rendimiento
/
Transcriptoma
Tipo de estudio:
Guideline
Límite:
Animals
Idioma:
En
Año:
2021
Tipo del documento:
Article