Evaluation of protocols for rRNA depletion-based RNA sequencing of nanogram inputs of mammalian total RNA


Autoři: Simon Haile aff001;  Richard D. Corbett aff001;  Steve Bilobram aff001;  Karen Mungall aff001;  Bruno M. Grande aff001;  Heather Kirk aff001;  Pawan Pandoh aff001;  Tina MacLeod aff001;  Helen McDonald aff001;  Miruna Bala aff001;  Robin J. Coope aff001;  Richard A. Moore aff001;  Andrew J. Mungall aff001;  Yongjun Zhao aff001;  Ryan D. Morin aff001;  Steven J. Jones aff001;  Marco A. Marra aff001
Působiště autorů: Canada’s Michael Smith Genome Sciences Centre, BC Cancer, Vancouver, British Columbia, Canada aff001;  Department of Molecular Biology and Biochemistry, Simon Fraser University, Burnaby, British Columbia, Canada aff002;  Department of Medical Genetics, University of British Columbia, Vancouver, British Columbia, Canada aff003
Vyšlo v časopise: PLoS ONE 14(10)
Kategorie: Research Article
doi: 10.1371/journal.pone.0224578

Souhrn

Next generation RNA-sequencing (RNA-seq) is a flexible approach that can be applied to a range of applications including global quantification of transcript expression, the characterization of RNA structure such as splicing patterns and profiling of expressed mutations. Many RNA-seq protocols require up to microgram levels of total RNA input amounts to generate high quality data, and thus remain impractical for the limited starting material amounts typically obtained from rare cell populations, such as those from early developmental stages or from laser micro-dissected clinical samples. Here, we present an assessment of the contemporary ribosomal RNA depletion-based protocols, and identify those that are suitable for inputs as low as 1–10 ng of intact total RNA and 100–500 ng of partially degraded RNA from formalin-fixed paraffin-embedded tissues.

Klíčová slova:

cDNA libraries – Messenger RNA – Mitochondria – Ribonucleases – Ribosomal RNA – RNA extraction – RNA sequencing – Sequence alignment


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Článek vyšel v časopise

PLOS One


2019 Číslo 10