The Nucleolus pp 231-241 | Cite as

Deep Sequencing Analysis of Nucleolar Small RNAs: RNA Isolation and Library Preparation

  • Baoyan Bai
  • Marikki LaihoEmail author
Part of the Methods in Molecular Biology book series (MIMB, volume 1455)


The nucleolus is a subcellular compartment with a key essential function in ribosome biogenesis. The nucleolus is rich in noncoding RNAs, mostly the ribosomal RNAs and small nucleolar RNAs. Surprisingly, also several miRNAs have been detected in the nucleolus, raising the question as to whether other small RNA species are present and functional in the nucleolus. We have developed a strategy for stepwise enrichment of nucleolar small RNAs from the total nucleolar RNA extracts and subsequent construction of nucleolar small RNA libraries which are suitable for deep sequencing. Our method successfully isolates the small RNA population from total RNAs and monitors the RNA quality in each step to ensure that small RNAs recovered represent the actual small RNA population in the nucleolus and not degradation products from larger RNAs. We have further applied this approach to characterize the distribution of small RNAs in different cellular compartments.

Key words

Nucleolus Small RNA purification Library preparation Deep sequencing 



The original work leading to the development of this protocol has been supported by NIH P30 CA006973 and Johns Hopkins University start-up funds.


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Copyright information

© Springer Science+Business Media New York 2016

Authors and Affiliations

  1. 1.Department of Immunology, Institute for Cancer Research, Oslo University Hospital, Oslo, and Centre for Cancer BiomedicineUniversity of OsloOsloNorway
  2. 2.Department of Radiation Oncology and Molecular Radiation SciencesJohns Hopkins University School of MedicineBaltimoreUSA
  3. 3.Sidney Kimmel Comprehensive Cancer CenterJohns Hopkins University School of MedicineBaltimoreUSA

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