Identification of Circular RNAs using RNA Sequencing

This protocol is the result of evaluating key aspects of circRNA library preparation, such as kit type, RNase R treatment, input amounts and their impact on circRNA detection. It enables optimal circRNA detection and provides data on adjustable parameters depending on the needs of the user. Identifying circRNAs in different sample types will help us to better understand the distribution of their expression and sets the stage for delineating the functional role of these molecules.

This method can be applied to any total RNA sample. It is important to keep RNA samples on ice prior to beginning the protocol. Assess the RIN and DV200 values on the Agilent Bioanalyzer or TapeStation before the prep, and follow appropriate procedures when working with RNA.

Begin by diluting total RNA to four micrograms in 39 microliters of RNase-free water and pipetting it up and down to mix. In a separate tube, use 1x RNase R Buffer to dilute the RNase R to a working concentration of two units per microliter. Pipette 39 microliters of total RNA and five microliters of 10x RNase R Reaction Buffer into a 1.5-microliter reaction tube, and mix.

Add six microliters of the diluted RNase R.Then adjust the pipette to full reaction volume, and mix the solution by pipetting up and down 10 times. Place the tube in a 37-degree Celsius water bath for 10 minutes, making sure that the full reaction volume is immersed in the water bath. Then, place the tube on ice, and immediately proceed with RNA cleanup and concentration.

Prior to starting, prepare the RNA Wash Buffer by mixing the concentrate with 48 milliliters of 100%ethanol, and place purification columns into collection tubes. When ready, add two volumes of RNA Binding Buffer to the RNase R-treated sample, and mix well. Add 150 microliters of 100%ethanol to the mixture for a total of 300 microliters, transfer the entire volume to the column, and centrifuge for 30 seconds.

Remove the flow through, and add 400 microliters of RNA Prep Buffer directly to the column. Centrifuge for 30 seconds, and discard the flow through. Then, add 700 microliters of RNA Wash Buffer to the column, centrifuge for 30 seconds, and discard the flow through.

Add another 400 microliters of the Wash Buffer, centrifuge for two minutes, and transfer the column to a fresh RNase-free 1.5-milliliter tube. Carefully add 11 microliters of RNase-free water directly to the column by holding the pipette tip right above the column filter. Let the sample sit for one minute at room temperature, and then centrifuge it for one minute.

Make sure there is flow through in the 1.5-milliliter tube, and discard the column. The purified RNA sample can be stored at minus 80 degrees Celsius or used immediately for library preparation. Transfer 10 microliters of the purified total RNA to a clean well in a 96-well PCR plate.

Add five microliters of rRNA Binding Buffer followed by five microliters of rRNA Removal Mix, then gently pipette up and down to mix the reagents. Seal the plate, and incubate it for five minutes at 68 degrees Celsius on a preprogrammed and preheated thermocycler block. After the incubation, let the plate sit at room temperature for one minute.

Remove the seal from the plate, and add 35 microliters of vortexed room temperature rRNA removal beads to the sample. Adjust the pipette to 45 microliters, and pipette up and down 10 to 20 times to thoroughly mix. Let the plate sit at room temperature for one minute.

Transfer the plate to a magnetic stand, and incubate it there for one minute or until the solution clears. Transfer the supernatant to new well on the plate. Then transfer the plate from the magnetic stand to the plate stand.

Vortex RNA cleanup beads until they are well dispersed, and add 99 microliters of beads to the sample. Pipette up and down to mix, and incubate the plate at room temperature for 10 minutes. Transfer the plate to the magnetic stand, and leave it there for five minutes or until the solution clears, then remove and discard all supernatant from the well.

With the plate still on the magnetic stand, add 200 microliters of freshly prepared 80%ethanol to the well without disrupting the beads. Leave the ethanol in the well for 30 seconds, then remove and discard it. Add 11 microliters of Elution Buffer to the well, and pipette it up and down to mix.

Incubate the plate at room temperature for two minutes, then transfer it back to the magnetic stand, and keep it there until the solution clears. Transfer 8.5 microliters of the supernatant to a new well on the same plate, and add 8.5 microliters of Elute, Primer, Fragment High Mix to each well containing a sample. Pipette up and down 10 times to mix thoroughly, seal the plate, and incubate it for eight minutes in a thermocycler preheated to 94 degrees Celsius.

Cool the sample to four degrees Celsius, remove the plate from the thermocycler, and centrifuge it briefly. Two library preparation kits, TruSeq and KAPA, were assessed for this protocol. Overall, a higher number of circular RNAs and lower percentage of ribosomal RNA was detected when using the TruSeq kit, so TruSeq was selected for further experiments.

The significance of RNase R pretreatment was evaluated by comparing the data generated from pretreated and non-pretreated libraries. As expected, a higher number of circular RNAs was consistently identified in the pretreated libraries compared to non-pretreated ones. The amount of input RNA was optimized for detecting a higher diversity of circular RNAs.

Libraries were prepared using one, two, and four micrograms of input RNA, and the highest diversity of circular RNA species was observed when using four micrograms of total RNA. The optimized protocol was used to compare circular RNA abundances in five brain regions from four healthy donors and for other tissue types from six healthy donors. Overall, a higher abundance of circular RNase was observed in the brain compared to other tissue types.

It is important to remember to follow appropriate procedures when working with RNA, including wearing gloves, thoroughly cleaning the bench and pipettes with RNase wipes or spray before beginning the protocol, and keeping RNA on ice beforehand. Following this procedure, orthogonal validation such as Sanger sequencing of identified circRNA junctions can be performed. The discovery of new circRNAs and further evidence of their presence carves out a new area of research for exploring their biological functions.