Computational protocol: Identification of senescence-associated circular RNAs (SAC-RNAs) reveals senescence suppressor CircPVT1

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Protocol publication

[…] After isolation of total RNA using TRIzol (Invitrogen) and assessment of quality and quantity using the Agilent 2100-Bioanalyzer, ribosomal RNA was removed by rRNA Depletion Nano kit (Qiagen) and 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), adaptors were ligated to cDNA using TruSeq ChIP Sample Preparation kit (Illumina, San Diego, CA, USA), the DNA fragments were size-selected (300–350 bp) after electrophoresis on a 2.5% agarose gel, and the selected DNA was subjected to 17 cycles of PCR amplification. Library quality was determined on the Bioanalyzer 2100 and the final libraries were sequenced using the Illumina HiSeq 2500 instrument. Data are available at GSE85771.For circRNA-Seq analysis, adapter contamination was removed from the raw FASTQ files and sequences were aligned to the human genome (hg19) with TopHat2 (v2.1.0), first to identify linear RNA and later to identify fusion transcripts using reads which did not align to the linear RNA. The program CIRCexplorer was run with the fusion transcripts obtained from TopHat2 using Ensembl GRCh37 Release 82 annotation to identify the circular RNAs (). Additionally, the cleaned FASTQ files (after removing adapters) were used for finding circularizing junctions by employing the find_circ software. In short, the reads were aligned to linear RNA using Bowtie2 program and then the unmapped reads were split into two anchors and aligned using Bowtie2 followed by identification of circularizing junctions (). Using CIRCexplorer and find_circ analysis, the combined circRNA junction read number from two proliferating and two senescent samples were normalized to the respective number of mapped reads and represented as ‘reads per million’. […]

Pipeline specifications

Software tools TopHat, CIRCexplorer, find_circ, Bowtie2
Application RNA-seq analysis
Organisms Homo sapiens