Rnase r

RPAD was performed according to the protocol as described in ref. ^{26 (link)}. In brief, 10 µg of RNA from nuclear and cytoplasmic RNA was incubated with RNase R (ABM, E049) for 30 min at 37 °C. Treated RNA was isolated using miRNeasy Mini Kit (Qiangen) following the manufacturer’s instructions and eluted with 30 µl of nuclease-free water. RNA was then incubated with polyA polymerase (NEB, M0276) for 30 min at 37 °C. 100 µl of Oligo-d(T)25 Magnetic Beads (NEB, S1419) were washed one time with lysis/binding buffer according to manufacturer’s instructions and were suspended in X2 lysis/binding buffer. RNA was incubated at 70 °C for 5 min, and was then incubated with the washed beads. Samples were incubated with shaking for 20 min. RNA samples with Oligo-dT beads were placed on a magnetic stand and the supernatant was collected. RNA was isolated using miRNeasy Mini Kit. Libraries for sequencing were generated using SENSE Total RNA-Seq Library Prep Kit (Lexogen, LX-042).

For the analyses of circRNA levels, total RNA was extracted from 200 µL of serum samples using Monarch Total RNA Miniprep Kit (New England, Ipswich, MS, USA). Extraction proceeded according to the manufacturer instructions. Minor modifications were applied including addition of a higher volume (500 µL) of isopropanol to each sample prior to loading to the purification column. DNAse I treatment was done according to the manufacturer recommendations. RNA elution with 40 µL of nuclease free water was preceded by additional centrifugation at 16,000 g/min RNAse R (10 U/µL) (ABM, Richmond, BC, Canada); treatment was performed to 10 µL of each sample in order to remove the linear RNA molecules prior to subsequent analyses of circRNAs.

5 ug of total RNA was incubated with or without 20 units of RNase R (Applied Biological Materials, Inc., catalog no. E049). Briefly, no (control) or 1.5 ul RNase R (30u) (test) and 3 ul of 10X RNase R buffer were added to 5 ug of RNA in a total volume of 30 ul and incubated in a 37C water bath for 30 minutes. Either no (control) or 1.5 ul (30 units) (test) more RNase R was added to reactions and incubated for an additional 1.5 hours in a 37C water bath. RNAs were cleaned and concentrated using the RNA Clean & Concentrator-5 kit (Zymo Research, catalog no. R1015) and eluted in 10ul H₂O for use in PCR reactions.

Total RNA samples containing circ_0000396 and its linear form solute carrier family 38 member 1 (SLC38A1) were treated with RNase R (100 μg/mL; Applied Biological Materials, Vancouver, Canada) for 20 min at 37 °C. Reverse transcription-quantitative polymerase chain reaction (RT-qPCR) was implemented to detect RNA levels.

Hsa_circ_0000190-containing samples were treated using RNase R (Applied BIOLOGICAL Materials, Vancouver, Canada) for 20 min at a dose of 100 μg/mL with subsequent qPCR analysis in order to confirm the circular nature of this transcript. To confirm the stability of this circRNA transcript, NSCLC cells were treated using Actinomycin D (Sigma) at a dose of 2 mg/mL, with transcript levels subsequently being analyzed via qPCR.

Ten microliters of RNase-free solution containing 10 μg of the total RNA of TE2 and TE5 cells was treated with RNase R (Applied Biological Materials, Richmond, BC, Canada). The RNase R mixture was combined with 2 μL of RNase R (20 U), 2 μL of 10× RNase R buffer, and 1 μL of RNase inhibitor (20 U) [Applied Biosystems]. Five microliters of RNase R mixture (test) or RNase-free water (control) was added to 10 µL of RNA solution in a total volume of 20 µL with RNase-free water and incubated at 37°C for 1 h. The products were then immediately placed on ice to stop the reaction and examined using the qRT-PCR assay as described above to measure linear and circFAT1 expression levels.