Selective Depletion of Abundant RNAs to Enable Transcriptome Analysis of Low‐Input and Highly Degraded Human RNA

Daniela B. Munafó1, Bradley W. Langhorst1, Christine L. Chater1, Christine J. Sumner1, Deyra N. Rodríguez1, Salvatore Russello1, Andrew F. Gardner1, Barton E. Slatko1, Fiona J. Stewart1, Dominick Sinicropi2, John Morlan2, Kunbin Qu2, Eileen T. Dimalanta1, Theodore B. Davis1

1 New England Biolabs, Ipswich, Massachusetts, 2 Genomic Health, Inc, Redwood City, California
Publication Name:  Current Protocols in Molecular Biology
Unit Number:  Unit 7.22
DOI:  10.1002/0471142727.mb0722s113
Online Posting Date:  January, 2016
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Ribosomal RNAs (rRNAs) are extremely abundant, often constituting 80% to 90% of total RNA. Since rRNA sequences are often not of interest in genomic RNA sequencing experiments, rRNAs can be removed from the sample before the library preparation step, in order to prevent the majority of the library and the majority of sequencing reads from being rRNA. Removal of rRNA can be especially challenging for low quality and formalin‐fixed paraffin‐embedded (FFPE) RNA samples due to the fragmented nature of these RNA molecules. The NEBNext rRNA Depletion Kit (Human/Mouse/Rat) depletes both cytoplasmic (5 S rRNA, 5.8 S rRNA, 18 S rRNA, and 28 S rRNA) and mitochondrial rRNA (12 S rRNA and 16 S rRNA) from total RNA preparations from human, mouse, and rat samples. Due to the high similarity among mammalian rRNA sequences, it is likely that rRNA depletion can also be achieved for other mammals but has not been empirically tested. This product is compatible with both intact and degraded RNA (e.g., FFPE RNA). The resulting rRNA‐depleted RNA is suitable for RNA‐seq, random‐primed cDNA synthesis, or other downstream RNA analysis applications. Regardless of the quality or amount of input RNA, this method efficiently removes rRNA, while retaining non‐coding and other non‐poly(A) RNAs. The NEBNext rRNA Depletion Kit thus provides a more complete picture of the transcript repertoire than oligo d(T) poly(A) mRNA enrichment methods. © 2016 by John Wiley & Sons, Inc.

Keywords: NGS; RNA depletion; sequencing; formalin‐fixed paraffin‐embedded; FFPE; RNA; rRNA; ribosomal RNA; library preparation; transcriptome; rRNA removal

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Table of Contents

  • Basic Protocol 1:  
  • Commentary
  • Figures
  • Tables
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Basic Protocol 1:  

  • RNA sample in nuclease‐free water (e.g., 0.01 to 1 μg total RNA; 12 μl total volume)
  • RNase H
  • 10× RNase H reaction buffer
  • NEBNext rRNA depletion solution
  • NEBNext probe hybridization buffer
  • DNase I (RNase‐free)
  • DNase I reaction buffer
  • Nuclease‐free water
  • 80% ethanol (prepared fresh; v/v pure ethanol with nuclease‐free water)
  • Agencourt RNAClean XP (Beckman Coulter, cat. no. A63987)
  • Magnetic rack (Alpaqua, cat. no. #A001322 or equivalent)
  • Thermal cycler (with heated lid)
NOTE: Starting material can be 0.01 to 1 μg of total RNA in 12 μl total volume. The RNA sample should be free of salts (e.g., Mg2+ or guanidinium salts), organics (e.g., phenol and ethanol) and chelating agents ethylene glycol‐bis(2‐aminoethylether)‐N,N,N′,N′‐tetraacetic acid (EGTA) or EDTA.NOTE: The RNA sample should be treated with DNase I to remove all traces of DNA. Remove DNase I by phenol/chloroform extraction and ethanol precipitation. DNase I contamination will reduce the depletion efficiency.NOTE: For RNA‐seq samples, the use of total RNA inputs >10 ng to increase library complexity (percentage of unique reads) and to reduce the number of duplicated reads (reads with identical sequence content) created by PCR is recommended.NOTE: This workflow is optimized for use with RNAs that have low RNA integrity number (RIN; Schroeder et al., ) values (RIN ≤7) such as FFPE RNA, as well as for use with high‐quality RNA (RIN >7).
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Literature Cited

Literature Cited
  Adiconis, X., Borges‐Rivera, D., Satija, R., DeLuca, D.S., Busby, M.A., Berlin, A.M., Sivachenko, A., Thompson, D.A., Wysoker, A., Fennell, T., Gnirke, A., Pochet, N., Regev, A., and Levin, J.Z. 2013. Comparative analysis of RNA sequencing methods for degraded or low‐input samples. Nat. Methods 10:623‐629. doi: 10.1038/nmeth.2483
  Morlan, J.D., Qu, K., and Sinicropi, D.V. 2012. Selective depletion of rRNA enables whole transcriptome profiling of archival fixed tissue. PLoS One 7:e42882. doi: 10.1371/journal.pone.0042882
  Schroeder, A., Mueller, O., Stocker, S., Salowsky, R., Leiber, M., Gassmann, M., Lightfoot, S., Menzel, W., Granzow, M., and Ragg, T. 2006. The RIN: An RNA integrity number for assigning integrity values to RNA measurements. BMC Mol. Biol. 7:3. doi: 10.1186/1471-2199-7-3
Internet Resources
  Web site for most recent updates to the NEBNext rRNA Depletion Kit (Human/Mouse/Rat).
  Web site for next‐generation sequencing library preparation reagents, protocols, and support.
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