Rnaclean xp magnetic beads

Transcriptome sequencing (RNA-seq) libraries were generated from 2 µg total RNA per replicate, using a published protocol (58 (link)) with minor modifications. Poly(A)⁺ RNA was purified using Dynabeads oligo(dT) (Life Technologies) according to the manufacturer’s protocol and fragmented by incubation at 94°C for 2 min to generate long fragments (>700 bp). cDNA was synthesized using Superscript II reverse transcriptase (Invitrogen) following the manufacturer’s protocol, and the resulting cDNA was purified using RNA Clean XP magnetic beads (Agencourt). Strand-specific libraries were generated with dUTP for second-strand synthesis. Double-stranded cDNA was end repaired, A-tailed, and ligated to adaptors as for the DNA library preparation (described above), and the resultant cDNA was purified and size selected to obtain 750-bp fragments. The uracil-containing second strand was then digested using uracil DNA glycosylase (Enzymatics), and cDNA was subjected to 15 cycles of PCR amplification using barcoded Illumina index primers (Table S2). The final cDNA was purified using AMPure XP beads (Agencourt) and eluted in 15 µl buffer EB (Qiagen). The concentration was determined by Qubit (as described above), yielding 6.33 to 37.5 ng RNA µl⁻¹, library quality was checked by Bioanalyzer, and equimolar pools were used for 150-bp paired-end sequencing on an Illumina HiSeq2500.

Total RNA was treated with DNase I, which degrades double-stranded and single-stranded DNA present in RNA samples. Ribosomal RNA was removed using the Ribo-off rRNA Depletion Kit (Vazyme, Inc.). Linear RNA was removed using RNase R (Epicenter, lnc.). Purification was performed using Agencourt RNAClean XP magnetic beads. The circular RNA molecules were fragmented and purified. Immediately following the RNA cleanup, first-strand cDNA synthesis was performed using directional RT buffer, followed by second-strand synthesis using the directional second strand buffer. End repaired and Y-Adapter ligation to A-tailed cDNA was followed by PCR. The amplified product was dissolved in Elution Buffer, and the library construction was completed. The library was qualified and quantitated by two methods: the distribution of the fragment size was checked using an Agilent 2100 bioanalyzer and the library was quantified using Qubit Fluorometer(Thermo Fisher Scientific, MA, USA). Finally, the qualified libraries were paired-end sequenced on MGISEQ-2000 (BGI-Shenzhen, China) platform with 100 bp read length.

Total RNAs were treated with DNase I and a Ribo-off rRNA Depletion Kit (Vazyme, Inc.) to degrade DNA and ribosomal RNA, respectively. Linear RNA was removed using RNase R (Epicentre, lnc). Purification was performed using Agencourt RNAClean XP magnetic beads. A tailing mix and RNA index adapters were added to perform end repair. The PCR products were denatured and circularized using the splint oligo sequence. Single-strand circular DNA was formatted as the final library. The library was checked for the distribution of fragments size using the Agilent 2100 bioanalyzer and quantified using BMG microplate reader (OMEGA). Finally, the qualified libraries were pair end sequenced on the BGISEQ-500 (BGI-Shenzhen, China). The software CIRI and find_circ is used to predict circRNA (Gao et al., 2015 (link); Memczak et al., 2013 (link)). All the datasets presented in this study were deposited in Genome Sequence Archive (GSA) repository with accession number PRJCA009585.

Caki-1 and Caki-1-R cells were lysed with Trizol reagent (Invitrogen, CA, USA) and RNA was isolated according to the standard method. RNA sequencing was performed by BGI Genomics Co., Ltd (Shenzhen, China). In total, 3 μg of total RNA was treated with DNase I to degrade DNA presenting in RNA samples. Then, ribosomal RNA was removed using the Ribo-off rRNA Depletion Kit and linear RNA was removed using RNase R. Purification was performed using Agencourt RNAClean XP magnetic beads. All other steps were performed according to the manufacturer’s protocols. The library was quality and quantitated in two methods: check the distribution of the fragments size using the Agilent 2100 bioanalyzer, and quantify the library using BMG (OMEGA). Finally, the Qualified libraries were sequenced pair end on the BGISEQ-500 or MGISEQ-2000 (BGI-Shenzhen, China). The RNA sequencing results are shown in Supplementary Table 5.

For circRNA-seq, total RNA was treated with RiboZero rRNA Removal Kit (Epicentre, WI, USA) to deplete ribosomal RNA (rRNA) following the manufacturer's protocols. The rRNA-depleted RNA samples were digested by RNase R to remove linear RNA followed by purification using Agencourt RNAClean XP magnetic beads. For mRNA-seq, total RNA was processed to enrich mRNAs with poly(A) tails by Oligo (dT) magnetic beads. The enriched circular RNA or mRNA samples were randomly fragmented into small pieces. DNA Libraries were prepared using TruSeq® Stranded kit and sequenced with HiSeq2500 (Illumina, San Diego, USA).