Protocol detail

Long-read sequencing of circularized RNA

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Total RNA (10 μg) was circularized with T4 RNA ligase 1⁸, digested with RNase R (Epicenter) to remove linear RNA, and termini were amplified with gene-specific primers listed in Supplementary Data 6. Two biological replicates of long-range single molecule real-time (SMRT) sequencing of 0.2–4 kb fragments was performed on a PacBio RS II system (Pacific Biosciences). Highly similar data sets were combined for final analysis. A single round of short-range sequencing was performed on a MiSeq instrument in 300 nt mode^{47 (link)}.

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Mesitov M.V., Yu T., Suematsu T., Sement F.M., Zhang L., Yu C., Huang L, & Aphasizheva I. (2019). Pentatricopeptide repeat poly(A) binding protein KPAF4 stabilizes mitochondrial mRNAs in Trypanosoma brucei. Nature Communications, 10, 146.

Publication 2019

RNase R PacBio RS II system

Biological Gene Primers Rna ligase Rnase r

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Variable analysis

independent variables

Circularization of total RNA with T4 RNA ligase 1
Digestion of linear RNA with RNase R
Amplification of termini with gene-specific primers

dependent variables

Long-range single molecule real-time (SMRT) sequencing of 0.2–4 kb fragments
Short-range sequencing on a MiSeq instrument in 300 nt mode

control variables

Two biological replicates for long-range SMRT sequencing

controls

Positive control: Not explicitly mentioned.
Negative control: Not explicitly mentioned.

Annotations

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