Ovation® RNA-Seq System V2

Protocol tips

Upstream tips	Protocol tips	Downstream tips
	Input Amounts 500 pg – 100 ng

Protocol tips
Input Amounts 500 pg – 100 ng

Publication protocol

After isolation of total RNA and RPAD-prepared RNA using miRNeasy Mini Kit, ribosomal (r)RNA was removed by rRNA Depletion Nano kit (Qiagen), whereupon cDNA was prepared and amplified using the Ovation RNA-Seq System V2 (NuGEN) kit following the manufacturer's instructions. The amplified cDNA was fragmented using a Bioruptor (Diagenode) followed by ligation of adaptors using TruSeq ChiP Sample Preparation kit (Illumina, San Diego, CA, USA), and DNA fragments ranging 300–350 bp were isolated from a 2.5% agarose gel and subjected to 17 cycles of PCR amplification. A Bioanalyzer 2100 instrument was used to analyze the quality of the cDNA libraries, which were sequenced using an Illumina HiSeq 2500 instrument (and deposited in GSE92632).
For identification of circRNAs in the RNA-Seq, adapter contamination was removed from the raw fastq files and TopHat2 (v2.1.0) was used to align the sequences to the human genome (hg19) and mouse genome (mm9). The aligned reads were used to identify the body of circular RNA and residual linear RNA using Cufflinks (v2.2.1) and default parameters (with the exception of assembled transfrags supported by five or more reported fragments). Both known (Ensembl GRCh37 Release 82 for human and Ensembl mm9 Release 67 for mouse) and novel transcripts were identified. The reads which did not align to the genome were used to identify fusion junctions using TopHat2. The CIRCexplorer program (v1.10) was used with the fusion junctions obtained from TopHat2 using the identified transcripts from the previous step to identify both the circularizing junction and the spliced sequence of circRNAs in HeLa and C2C12 cells,

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Manufacturer protocol

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