Optimized design of antisense oligomers for targeted rRNA depletion (Phelps et al 2020))
Protocol for RNaseH-mediated RNA depletion
Optimized design of antisense oligomers for targeted rRNA depletion
Wesley A. Phelps, Anne E. Carlson, Miler T. Lee, 2020. []
This protocol describes how to deplete abundant RNA species (e.g., ribosomal RNA) from total RNA using the method described in Phelps et al, 2020. Antisense DNA oligos that sparsely tile target RNA sequences are designed using Oligo-ASST, https://mtleelab.pitt.edu/oligo , and used in an RNaseH digestion reaction followed by column clean up. Depleted RNA is suitable for downstream applications such as RNA-seq and qRT-PCR.
Thermal cycler Centrifuge Pipets to measure 1 to 700 μL Consumables: PCR tubes and caps compatible with the thermal cycler Aerosol filter pipet tips 1.5 mL tubes
5x hybridization buffer (500mM Tris-HCl pH 7.4, 1M NaCl, 50mM DTT) Nuclease-free water 100% ethanol RNase-Away decontamination reagent (Invitrogen #10328011) Zymo Clean and Concentrator-5 kit (#D4013) NEB Thermostable RNaseH enzyme and 10x buffer (#M0523S) TURBO DNase enzyme and 10x buffer (Invitrogen #AM2238)
Antisense DNA oligos targeting RNAs to be depleted (e.g., rRNAs) Purified total RNA
Oligo design (once per target RNA)
1. Curate the target RNA sequences using Web databases such as Genbank, the UCSC Genome Browser, or Ensembl. For eukaryotic rRNA depletion, you would typically include the three nuclear rDNA sequences (e.g., 28S, 18S and 5.8S) and the two mitochondrial rDNA sequences (16S and 12S).
2. Design antisense oligos using the Oligo-ASST Web tool, https://mtleelab.pitt.edu/oligo . Input your sequences one at a time or all at once in a single FASTA file and click the “Calculate” button to use the default settings (39-40 nt oligos spaced ≤30 nts apart, with melting temperature as close to 70-80oC as possible). Download the oligo sequences to your computer using the “Export” button and open the file using Microsoft Excel or similar program.
3. Order the oligos at the desired scale and standard desalting – 25 nmol scale per oligo should be sufficient for >1000 nuclear rRNA depletion reactions from 1μg of total RNA. Some vendors offer bulk/value pricing when ordering above a threshold number of oligos.
(Warning: if you downloaded the Details file from Oligo-ASST, be sure to order the antisense oligos and *NOT* the target sequences).
All remaining steps should be done in conditions that minimize contamination with unwanted RNases. Wipe all surfaces and tools with RNase-Away and use filter tips and clean consumables.
Constructing oligo pools
4. Resuspend each dry oligo tube to 1000 μM with nuclease-free water.
5. Create a 10x working stock of oligos by combining 1μL of each oligo into a tube, then diluting with nuclease-free water to the desired concentration per oligo. For eukaryotes, a nuclear rRNA 10x stock should be 4 μM per individual oligo, to be about 10-fold in excess of the target RNA species in 1μg of total RNA in a 10 μL hybridization reaction. A 10x stock at 1μM per oligo may be sufficient for a mitochondrial rRNA pool, depending on taxon and cellular context.
Oligo hybridization to RNA
6. Combine 1 μL of each 10x oligo pool (e.g., nuclear rRNA pool + mitochondrial rRNA pool) with 1 μg of total RNA in a PCR tube.
7. Add 2 μL of 5x hybridization buffer and bring the reaction to 10 μL with nuclease-free water.
8. Mix well by pipetting and place the capped tube in the thermocycler with the following hybridization program: a. 95°C for 2 minutes (this facilitates RNA denaturation) b. Cool the reaction to 22°C at a rate of 0.1°C/s and hold at 22°C for 5 minutes
9. Place the reaction on ice.
10. Add 10U of thermostable RNaseH and 2μL of 10x RNaseH buffer to the reaction. Bring the reaction volume to 20μL with nuclease-free water.
11. Mix well by pipetting and heat the depletion reaction at 65°C for 5 minutes or 45oC for 30 minutes. Reaction temperature should be near or above the predicted melting temperatures of the oligos.
12. Place the reaction on ice.
13. Add 5U of TURBO DNase and 5μL of 10x DNase buffer. Bring the reaction volume to 50μL with nuclease-free water.
14. Mix well by pipetting and heat the reaction at 37°C for 30 minutes.
15. Place the reaction on ice.
Clean-up and size selection
Follow the manufacturer’s protocol for the Zymo Clean and Concentrator-5 kit to enrich for RNA molecules >200 nts:
16. Mix 50 μL of Zymo RNA Binding Buffer and 50 μL of 100% ethanol to create the Adjusted Binding Buffer.
17. Add 100 μL (2 volumes) of Adjusted Binding Buffer to depleted sample, which should be in a volume of 50μL after Step 15. Mix well by pipetting.
18. Transfer the sample to a Zymo spin column with collection tube. Centrifuge for 30 seconds at 15,000 x g.
19. Discard the flow through, which will contain RNAs < 200 nts
20. Add 400 μL of RNA Prep Buffer to the column. Centrifuge for 30 seconds at 15,000 x g. Discard flow through.
21. Add 700 μL of RNA Wash Buffer to the column. Centrifuge for 30 seconds at 15,000 x g. Discard flow through.
22. Add 400 μL of RNA Wash Buffer to the column. Centrifuge for 1 minute at 15,000 x g. Discard flow through.
23. Place the column into a nuclease-free 1.5 mL microcentrifuge tube for elution.
24. Add 6 μL of nuclease-free water directly to the column matrix and centrifuge for 30 seconds at 15,000 x g.
25. Proceed straight to downstream application (e.g., RNA-seq library building) or store purified RNA at -80°C until needed.