GSM5396518: PMY_Frac12; synthetic construct; RNA-Seq
IRES screening: Total DNA from each expression bin was extracted using Quick-DNA Microprep Plus Kit (Zymo Research).Synthesis (NEB BioLabs), and purified with DNase digestion (TURBO DNase; Thermo Fisher Scientific) and RNase R digestion (MACLAB; 20 U RNase R per 20 μg of RNA). Polysome profiling: The total RNAs of each fraction were purified respectively using RNeasy Mini Kit (Qiagen) with DNase treatment (Qiagen). The RNAs were then treated with RNase R and purified using RNA Clean & Concentrator-25 (Zymo Research). M2-seq: The circRNA of oligo of interest was produced with in vitro transcription using HiScribe™ T7 High Yield RNA IRES screening: Three rounds of PCR using NEBNext Ultra II Q5 Master Mix (NEB BioLabs) were performed to generate the library for next-generation sequencing. For the first PCR, oligo library sequence was amplified using the following primer set – Fw: GGGATCACTCTCGGCATGGA; Rv: GCTCCTCGCCCTTGCTCAC. Each 50 μL PCR reaction contained 200 ng total DNA, 500 nM forward primer, and 500 nM reverse primer. The parameters for the PCR were 98°C for 1 min, 24 cycles of 98°C for 30 s, 65°C for 30 s, 72°C for 40 s, each, and finally one cycle of 72°C for 5 min. For the second PCR, adapter priming sequences were added to the oligo library using the following primer set – Fw: TCGTCGGCAGCGTCAGATGTGTATAAGAGACAGGGGATCACTCTCGGCATGGA; Rv: GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAGGCTCCTCGCCCTTGCTCAC (underlined sequence represents the sequence for illumina adapters priming). Each 50 μL PCR reaction contained 1.25 μL of the first PCR product, 500 nM forward primer, and 500 nM reverse primer. The parameters for the PCR were 98°C for 1 min, 18 cycles of 98°C for 30 s, 70°C for 30 s, 72°C for 40 s, each, and finally one cycle of 72°C for 5 min. For the third PCR, custom barcodes adapters were used for the reaction. Each 50 μL PCR reaction contained 1 μL of 1:20 diluted second PCR product, 1 μM Ad1 adapter, and 1 μM Ad2 adapter. The parameters for the PCR were 98°C for 1 min, 15 cycles of 98°C for 30 s, 72°C for 30 s, 72°C for 40 s, each, and finally one cycle of 72°C for 5 min. The PCR product was then separated on a 2% E-Gel™ EX Agarose Gel (Invitrogen) and the DNA fragments with expected size were purified using Zymoclean Gel DNA Recovery Kit (Zymo Research). Polysome profiling: The oligo sequences from each sample were amplified by RT-PCR with SuperScript™ IV One-Step RT-PCR System (Thermo Fisher Scientific) using primers flanking the oligo sequence (Fw: GGGATCACTCTCGGCATGGA; Rv: GCTCCTCGCCCTTGCTCAC). cDNA libraries were generated and sequenced with HiSeq 4000 using the library generation method as for IRES screening. M2-seq: The complimentary single-stranded DNA of the circRNA was generated by reverse transcription using SuperScript™ IV (Thermo Fisher Scientific) with oligo sequence specific primer (TCTTTTGTTGGAGTCTACTCGACTCCTTT) and purified with DNA Clean & Concentrator-5 (Zymo Research). Three rounds of PCR using NEBNext Ultra II Q5 Master Mix (NEB BioLabs) were performed to generate the cDNA library for next-generation sequencing. For the first PCR, oligo library sequence was amplified using the following primer set – Fw: GAACGACTCGAGTAGAGTCGAAAA; Rv: TCTTTTGTTGGAGTCTACTCGACTCCTTT. Each 50 μL PCR reaction contained 200 ng total DNA, 500 nM forward primer, and 500 nM reverse primer. The parameters for the PCR were 98°C for 1 min, 15 cycles of 98°C for 30 s, 67°C for 30 s, 72°C for 40 s, each, and finally one cycle of 72°C for 5 min. For the second PCR, sequences for adapters priming were added to the oligo library using the following primer set – Fw: TCGTCGGCAGCGTCAGATGTGTATAAGAGACAGGAACGACTCGAGTAGAGTCGAAAA; Rv: GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAGTCTTTTGTTGGAGTCTACTCGACTCCTTT (underline represents the sequence for illumina adapters priming). Each 50 μL PCR reaction contained 1.25 μL of the first PCR product, 500 nM forward primer, and 500 nM reverse primer. The parameters for the PCR were 98°C for 1 min, 12 cycles of 98°C for 30 s, 67°C for 30 s, 72°C for 40 s, each, and finally one cycle of 72°C for 5 min. For the third PCR, custom barcodes adapters were used for the reaction. Each 50 μL PCR reaction contained 1 μL of 1:20 diluted second PCR product, 1 μM Ad1 adapter, and 1 μM Ad2 adapter. The parameters for the PCR were 98°C for 1 min, 15 cycles of 98°C for 30 s, 72°C for 30 s, 72°C for 40 s, each, and finally one cycle of 72°C for 5 min. The PCR product was then separated on a 2% E-Gel™ EX Agarose Gel (Invitrogen) and the DNA fragments with expected size were purified using Zymoclean Gel DNA Recovery Kit (Zymo Research). IRES screening: DNA-seq. Polysome profiling: RNA-seq M2-seq: RNA-seq