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 conserved²

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 ad....

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

Name	Company	Catalog Number	Comments
1000 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