Increasing quality, throughput and speed of sample preparation for strand-specific messenger RNA sequencing.
BMC Genomics
; 18(1): 515, 2017 07 05.
Article
en En
| MEDLINE
| ID: mdl-28679365
ABSTRACT
BACKGROUND:
RNA-Sequencing (RNA-seq) is now commonly used to reveal quantitative spatiotemporal snapshots of the transcriptome, the structures of transcripts (splice variants and fusions) and landscapes of expressed mutations. However, standard approaches for library construction typically require relatively high amounts of input RNA, are labor intensive, and are time consuming. METHODS Here, we report the outcome of a systematic effort to optimize and streamline steps in strand-specific RNA-seq library construction. RESULTS This work has resulted in the identification of an optimized messenger RNA isolation protocol, a potent reverse transcriptase for cDNA synthesis, and an efficient chemistry and a simplified formulation of library construction reagents. We also present an optimization of bead-based purification and size selection designed to maximize the recovery of cDNA fragments.CONCLUSIONS:
These developments have allowed us to assemble a rapid high throughput pipeline that produces high quality data from amounts of total RNA as low as 25 ng. While the focus of this study is on RNA-seq sample preparation, some of these developments are also relevant to other next-generation sequencing library types.Palabras clave
Texto completo:
1
Banco de datos:
MEDLINE
Asunto principal:
Manejo de Especímenes
/
ARN Mensajero
/
Biblioteca de Genes
/
Análisis de Secuencia de ARN
Tipo de estudio:
Prognostic_studies
Límite:
Humans
Idioma:
En
Año:
2017
Tipo del documento:
Article