Identification of Circular RNAs using RNA Sequencing

Summary

Circular RNAs (circRNAs) are non-coding RNAs that may have roles in transcriptional regulation and mediating interactions between proteins. Following assessment of different parameters for construction of circRNA sequencing libraries, a protocol was compiled utilizing stranded total RNA library preparation with RNase R pre-treatment and is presented here.

Abstract

Circular RNAs (circRNAs) are a class of non-coding RNAs involved in functions including micro-RNA (miRNA) regulation, mediation of protein-protein interactions, and regulation of parental gene transcription. In classical next generation RNA sequencing (RNA-seq), circRNAs are typically overlooked as a result of poly-A selection during construction of mRNA libraries, or are found at very low abundance, and are therefore difficult to isolate and detect. Here, a circRNA library construction protocol was optimized by comparing library preparation kits, pre-treatment options and various total RNA input amounts. Two commercially available whole transcriptome library preparation kits, with and without RNase R pre-treatment, and using variable amounts of total RNA input (1 to 4 µg), were tested. Lastly, multiple tissue types; including liver, lung, lymph node, and pancreas; as well as multiple brain regions; including the cerebellum, inferior parietal lobe, middle temporal gyrus, occipital cortex, and superior frontal gyrus; were compared to evaluate circRNA abundance across tissue types. Analysis of the generated RNA-seq data using six different circRNA detection tools (find_circ, CIRI, Mapsplice, KNIFE, DCC, and CIRCexplorer) revealed that a stranded total RNA library preparation kit with RNase R pre-treatment and 4 µg RNA input is the optimal method for identifying the highest relative number of circRNAs. Consistent with previous findings, the highest enrichment of circRNAs was observed in brain tissues compared to other tissue types.

Introduction

Circular RNAs (CircRNAs) are endogenous, non-coding RNAs that have gained attention given their pervasive expression in the eukaryotic transcriptome^1,2,3. They are formed when exons back-splice to each other and hence were initially considered to be splicing artifacts^4,5. However, recent studies have demonstrated that circRNAs exhibit cell type, tissue, and developmental stage specific expression^3,6 and are evolutionarily conserved^2,3. Furthermore, they are involved in mediation of protein-protein interactions⁷, micro-RNA (miRNA) binding^3,8,9,10, and regulation of parental gene transcription¹¹.

In classical RNA sequencing (RNA-seq), circRNAs may be completely lost during library construction as a result of poly-A selection for mRNAs or may be difficult to isolate given their low abundance. However, recent circRNA characterization studies have incorporated a pre-treatment step using RNase R in order to enrich for circRNAs^2,12,13. RNase R is an exoribonuclease that digests linear RNAs, leaving behind circular RNA structures. CircRNA enrichment protocols were optimized by generating and comparing data from two commercially available whole transcriptome library construction kits, with and without an RNase R pre-treatment step, and using varying amounts of total RNA input (1 to 4 µg). The optimized protocol was next used to evaluate the abundance of circRNAs across five different brain regions (cerebellum [BC], inferior parietal lobe [IP], middle temporal gyrus [MG], occipital cortex [OC] and superior frontal gyrus [SF]) and four other tissue types (liver [LV], lung [LU], lymph node [LN] and pancreas [PA]). RNA-seq libraries were paired end sequenced and data was analyzed using six different circRNA prediction algorithms: find_circ³, CIRI¹⁴, Mapsplice¹⁵, KNIFE¹⁶, DCC¹⁷, and CIRCexplorer¹⁸. Based on our analysis, the highest number of unique circRNAs was detected when using a stranded total RNA library preparation kit with RNase R pre-treatment and 4 µg total input RNA. The optimized protocol is described here. As previously reported^19,20, the highest enrichment of circRNAs was observed in the brain compared to other tissue types.

Protocol

This research has been performed in compliance with all institutional, national and international guidelines for human welfare. Brain tissues were obtained from the Banner Sun Health Research Institute Brain and Body Donation Program in Sun City, AZ. The operations of the Brain and Body Donation Program are approved by the Western Institutional Review Board (WIRB protocol #20120821). All subjects or their legal representatives signed the informed consent. Commercial (non-brain) biospecimens were purchased from Proteogenex.

1. RNase R Treatment

NOTE: In the following steps, the reaction volume is adjusted to a total volume of 50 µL. This is the minimum sample volume to be used in the RNA cleanup & concentrator kit (see Table of Materials). Additionally, the optimized protocol described here is for an input amount of 4 µg of total RNA. A longer incubation time for RNase R treatment is recommended for an input amount >4 µg.

1. Dilute total RNA to 4 µg in 39 µL RNase-free water in a microcentrifuge tube and mix well by pipetting.
2. In a separate tube, dilute the RNase R to a working concentration of 2 U/µL with 1x RNase R Reaction Buffer. Make only enough for immediate use.
3. Pipette 39 µL of total RNA and 5 µL of 10x RNase R Reaction Buffer into a 1.5 mL reaction tube and mix well by pipetting (50 µL will be the total reaction volume). Next, add 6 µL of RNase R (2 U/µL).
4. Adjust the pipette to the full reaction volume (50 µL) and mix well by pipetting up and down 10 times.
5. Place the tube in a 37 °C water bath for 10 min. Make sure that the full reaction volume is immersed in the water bath.
6. Place the tube on ice and immediately proceed with RNA cleanup & concentration (section 2).

2. Purifying RNA Using an RNA Cleanup and Concentrator Kit

NOTE: When using high quality RNA (RIN>8, DV200>80%), RNase R treatment may result in loss of approximately 60% of RNA. Using a 4 µg input, it is estimated that 2–2.5 µg of treated RNA is left after section 1.

1. Before starting, prepare the RNA Wash Buffer by adding 48 mL of 100% ethanol to the buffer concentrate and mix well by pipetting. Place purification columns into collection tubes (see Table of Materials) and place in a tube rack.
   NOTE: Use the following centrifugation settings for all following steps: 10,000–16,000 x g. If DNase I treatment has already been performed, skip DNase I treatment at this stage.
2. Add 2 volumes of RNA Binding Buffer to the RNase R treated sample, and mix well by pipetting (total volume: 150 µL).
3. Add 1 volume of 100% ethanol to the RNA Binding Buffer and RNase R treated sample mixture, and mix well by pipetting (total volume: 300 µL).
4. Transfer the entire volume to the column and centrifuge the column for 30 s. Discard flow through.
5. Add 400 µL of RNA Prep Buffer directly to the column, centrifuge the column for 30 s, and discard flow through.
6. Add 700 µL of RNA Wash Buffer directly to the column, centrifuge the column for 30 s, and discard flow through.
7. Add 400 µL of RNA Wash Buffer directly to the column, centrifuge the column for 2 min, and transfer the column to a fresh RNase-free 1.5 mL tube.
8. Add 11 µL of RNase-free water directly to the column by holding the pipette tip right above the column filter and ensuring that water lands only on the column filter.
9. Incubate the column for 1 min at room temperature and centrifuge for 1 min.
10. Before discarding the column, check for flow through in the RNase-free tube. If elution was successful, store sample at -80 °C or immediately proceed with library preparation. The final total elution volume of approximately 10 µL is used for library construction.
    NOTE: Stopping point: Leave RNA at -80 °C for up to 7 days before continuing with library preparation.

3. circRNA Library Prep

NOTE: See Table of Materials for kit, which contains most reagents used in this section.

1. rRNA depletion and fragmentation
   1. Transfer 10 µL of purified RNA from step 2.10 to a clean well in a new 96-well 0.3 mL PCR plate. To the well, add 5 µL of rRNA Binding Buffer followed by 5 µL rRNA Removal Mix. Gently pipette up and down 10 times to mix.
   2. Seal plate and incubate for 5 min at 68 °C on a pre-programmed, pre-heated thermocycler block. After completion of the 5 min incubation, place plate on bench and incubate at room temperature for 1 min.
   3. Remove seal from plate. Add 35 µL of vortexed room temperature rRNA removal beads to sample. Adjust the pipette to 45 µL and pipette up and down 10–20x to mix thoroughly. Incubate plate for 1 min at room temperature.
   4. Transfer plate to a magnetic stand and incubate on the stand for 1 min or until the solution clears. Transfer all of the supernatant (~45 µL) to new well on the same plate, or new plate (depending on how many samples you are working with).
   5. Vortex the RNA cleanup beads (see Table of Materials) until well dispersed, and add 99 µL of beads to each sample. Pipette up and down 10x to mix. Incubate the plate at room temperature for 10 min.
   6. Transfer the plate to the magnetic stand and incubate an additional 5 min or until the solution clears. Remove and discard all of the supernatant from the well.
   7. With the plate still on the magnetic stand, add 200 µL of freshly prepared 80% EtOH to the well without disrupting the beads. Incubate for 30 s, then remove and discard ethanol. Repeat for a total of 2 washes.
   8. Add 11 µL of Elution Buffer to each well and pipette up and down 10 times to mix. Incubate at room temperature for 2 min, and then transfer to the magnetic stand until the solution clears (1–5 min).
   9. Transfer 8.5 µL of the supernatant from the well to a new well on the same plate or to a new plate. Add 8.5 µL of the Elute, Primer, Fragment High mix to each well containing sample. Pipette up and down 10 times to mix thoroughly.
   10. Seal plate and incubate for 8 min at 94 °C on a pre-programmed, pre-heated thermocycler block. Remove from thermocycler when it reaches 4 °C and centrifuge briefly.
       NOTE: Proceed immediately to the Synthesize First Strand cDNA protocol.
2. Synthesize cDNA
   1. For each sample being prepared, mix 9 µL First Strand Synthesis Mix with 1 µL of reverse transcriptase (see Table of Materials). Add 8 µL of the mixture to the sample. Pipette up and down 6 times to mix.
      1. Seal plate and incubate on a pre-programmed, pre-heated thermocycler block using the following parameters: 25 °C for 10 min, 42 °C for 15 min, 70 °C for 15 min, 4 °C hold. Proceed immediately to second strand synthesis.
   2. Add 5 µL of Resuspension buffer to each sample followed by 20 µL of Second Strand Marking master mix. Pipette the entire volume up and down 6 times.
   3. Seal plate and incubate on a pre-programmed, pre-heated thermocycler block set to 16 °C for 1 h. Following incubation, remove the plate from the thermocycler and let it equilibrate to room temperature.
   4. Vortex PCR purification beads (see Table of Materials) and add 90 µL beads to each well of sample. Pipette up and down 10 times to mix thoroughly. Incubate at room temperature for 10 min.
   5. Transfer the bead/sample mix to the magnetic stand and incubate for 5 min or until liquid clears. Remove and discard supernatant.
   6. Add 200 µL of 80% EtOH to each sample. Incubate samples on the magnetic stand at room temperature for 30 s. Discard supernatant. Repeat 1x.
   7. Allow beads to dry at room temperature for 6 min, and then remove from magnetic stand.
   8. Resuspend beads in 19.5 µL of Resuspension buffer. Pipette up and down 10 times to mix thoroughly. Incubate at room temperature for 2 min, then transfer to the magnetic stand and incubate for an additional 1 min or until the liquid clears.
   9. Transfer 17.5 µL of supernatant to new well/new plate.
      NOTE: If not proceeding immediately, samples can be stored at -20 °C for up to 7 days.
3. Library preparation
   1. Add 12.5 µL of A-Tailing Mix to each well containing supernatant. Pipette the entire volume up and down 10 times to mix.
   2. Incubate the reaction on a pre-programmed, pre-heated thermocycler block set to 37 °C using the following parameters: 37 °C for 30 min, 70 °C for 5 min, 4 °C hold. When samples reach 4 °C, proceed immediately to adapter ligation.
   3. To each sample add 2.5 µL of Resuspension buffer, 2.5 µL of a unique RNA adapter, and 2.5 µL of Ligation Mix. Pipette up and down 10x to mix.
   4. Incubate samples on a pre-programmed, pre-heated thermocycler block at 30 °C for 10 min.
   5. Add 5 µL of Stop Ligation buffer to each sample and pipette up and down to mix.
   6. Add 42 µL of mixed PCR purification beads to each sample and mix thoroughly. Follow steps 3.2.6 through 3.2.10, but change the resuspension volume to 52 µL and final elution volume to 50 µL.
   7. Repeat the PCR purification bead protocol again with the 50 µL elution from step 3.3.6, but change the resuspension volume to 22 µL and final elution volume to 20 µL.
      NOTE: If not proceeding immediately, samples can be stored at -20 °C for up to 7 days.
   8. Add 5 µL of PCR Primer Cocktail and 25 µL of PCR Master Mix to each sample. Mix by pipetting up and down 10 times. Incubate the reaction on a pre-programmed, pre-heated thermocycler block using the following parameters: 98 °C for 30 s; then 8 cycles of 98 °C for 10 s, 60 °C for 30 s, and 72 °C for 30 s; then 72 °C for 5 min, then 4 °C hold.
      NOTE: Optimization of the total number of PCR cycles may be needed to generate sufficient amounts of library for sequencing.
   9. Follow the protocol for PCR bead purification (steps 3.2.4 through 3.2.9), except add 50 µL of well-mixed PCR purification beads and change the resuspension volume to 32.5 µL with a final elution volume of 30 µL.
      NOTE: Samples should be stored at -20 °C.
4. Quantification and Quality Control using a nucleic acid analyzer
   1. Allow tapes and reagents to equilibrate at room temperature for 30 min.
   2. Mix 2 µL of library with 2 µL of HS D1000 buffer, and add to a compatible well plate.
   3. Seal tightly with compatible foil seal, and vortex for 1 min at 2,000 rpm.
   4. Spin down and load plate on the analyzer following software prompts.
      NOTE: Libraries should be approximately 260 bp in size.

4. Data Analysis Workflow

1. Sequence RNA-seq libraries (see Table of Materials) to generate 82 bp paired-end reads. Convert raw sequencing data in the form of basecall files (.bcl) to FASTQs using the bcl2fastq tool (v0.2.19).
2. Detect circRNAs.
   NOTE: Based on previously reported evidence that an ensemble circRNA detection approach performs better compared to using a single detection tool^21,22, we suggest using multiple tools for circRNA detection. Here, circRNAs were identified using six existing circRNA prediction algorithms: find_circ, CIRI, CIRCexplorer, Mapsplice, KNIFE, and DCC, applying the recommended parameter settings for each algorithm.
   1. Download and install each circRNA detection algorithm on a Linux high performance computing cluster using the instructions provided by the developers.
   2. Align RNA-seq FASTQs against the reference genome (GRCh37), utilizing the aligner recommended for each tool.
   3. Following alignment, execute circRNA detection algorithms by applying their respective recommended parameter settings.
   4. Each tool will output a multi-column results file with the list of detected circRNAs, extract the circRNA co-ordinates and the number of supporting reads from this in order to quantify the number of candidates detected in each sample/test condition.
3. Convert the circRNA coordinates output by CIRI, Mapsplice, and DCC to 0-based coordinates to be consistent with the other three algorithms.
4. Select circRNAs with two or more supporting reads or downstream analyses and comparisons. Table 1 summarizes all the parameters evaluated in our study along with the total number of sequencing reads generated for each sample.
5. For each sample/test condition, count the number of detected circRNAs normalized to the number of mapped reads generated for that library, per million. Summarize the results across the various tools/samples in box plots, as detailed in the Representative Results.

Results

Data generated using a commercially available universal control RNA (UC) and using two library preparation kits, both of which include a ribo-depletion step in their protocols, was first assessed. Using an analytical workflow (Data analysis workflow, section 4), overall, a higher number of circRNAs was detected in the TruSeq datasets compared to the Kapa ones (Figure 1). Although the ribosomal RNA (rRNA) percentages were below 5% in datasets from both kits for lower input amounts (1, 2 ug), ...

Discussion

In this study, two commercially available library preparation kits, pre-treatment options, and input RNA amounts were tested in order to optimize a circRNA enrichment protocol for construction of circRNA sequencing libraries. Based on this study’s assessments, a number of key aspects and critical steps in creating circRNA sequencing libraries are apparent. Our evaluation confirms the utility of RNase R pre-treatment, as reflected by the increased number of circRNAs detected. Overall, a higher diversity of circRNAs ...

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
1,000 µL pipette tips	Rainin	GP-L1000F
20 µL pipette tips	Rainin	SR L 10F
200 µL pipette tips	Rainin	SR L 200F
2200 TapeStation Accessories (foil covers)	Agilent Technologies	5067-5154
2200 TapeStation Accessories (tips)	Agilent Technologies	5067-5153
Adhesive Film for Microplates	VWR	60941-064
AMPure XP Beads 450 mL	Beckman Coulter	A63882	PCR purification
Eppendorf twin.tec 96-Well PCR Plates	VWR	951020401
High Sensitivity D1000 reagents	Agilent Technologies	5067-5585
High Sensitivity D1000 ScreenTape	Agilent Technologies	5067-5584
HiSeq 2500 Sequencing System	Illumina	SY-401-2501
HiSeq 3000/4000 PE Cluster Kit	Illumina	PE-410-1001
HiSeq 3000/4000 SBS Kit (150 cycles)	Illumina	FC-410-1002
HiSeq 4000 Sequencing System	Illumina	SY-401-4001
HiSeq PE PE Rapid Cluster Kit v2	Illumina	PE-402-4002
HiSeq Rapid SBS Kit v2 (50 cycle)	Illumina	FC-402-4022
Kapa Total RNA Kit	Roche	KK8400
Molecular biology grade ethanol	Fisher Scientific	BP28184
Qubit Assay Tubes	Supply Center by Thermo Fischer	Q32856
Qubit dsDNA High Sense Assay Kit	Supply Center by Thermo Fischer	Q32854
RNA cleanup and concentrator - 5	Zymo	RCC-100	Contains purification columns, collection tubes
RNAClean XP beads	Beckman Coulter Genomics		RNA Cleanup beads
Rnase R	Lucigen	RNR07250
SuperScript II Reverse Transcriptase 10,000 units	ThermoFisher (LifeTech)	18064014
TapeStation 2200	Agilent Technologies		Nucleic Acid analyzer
TElowE	VWR	10128-588
TruSeq Stranded Total RNA Library Prep Kit	Illumina	20020596	Kit used in section 3
Two-Compartment Divided Tray	VWR	3054-1004
UltraPure Water	Supply Center by Thermo Fischer	10977-015
Universal control RNA	Agilent	740000