Computational protocol: Genome-Wide Identification of Circular RNAs in Arabidopsis thaliana

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Protocol publication

[…] We used the NGS QC Toolkit v2.3.3 software to filter the reads with adapter sequences, reads with an unknown base (N, unknown bases in a read > 5%), and low-quality reads (reads with base quality ≤ 20). Sequence alignments were performed using TopHat2 with BOWTIE2 (v2.0.5) () software for mapping to the reference genome (Arabidopsis_thaliana TAIR10.30 ENSEMBL). The circRNAs were predicted using the find_circ program with the same parameters as described previously (). Chimeric mapped reads were selected for circular (“back-spliced”) junctions if the sequence reads mapped to one chromosome on the same strand, while the two sequence segments mapped to the genomic region with reverse order. CircRNA abundance was predicted on the basis of circular junction read counts. The overlapping region between predicted circRNAs and the gene region of the A. thaliana reference genome was identified using Bedtools software (), and the circRNAs with overlapping regions were annotated. All expression data and other information (coordinates, strand, etc.) of identified A. thaliana circRNAs are in GSE77672. To evaluate the conservation of circRNAs, the circRNA sequences of A. thaliana, O. sativa, H. vulgare, S. lycopersicum, and Z. mays were first downloaded from PlantcircBase. Then, our identified circRNA sequences (Supplementary Table ) from A. thaliana were used for a BLAST search (BlastN in BLAST+, v2.2.27, E < 1e-5) against the circRNA sequences from A. thaliana, O. sativa, H. vulgare, S. lycopersicum, and Z. mays.To evaluate the potential functions of the parent genes of circRNAs, the parent mRNAs were used for BLAST searches and functionally categorized according to gene ontology (GO) annotation by BLAST2GO software with the default parameters (). To predict miRNA–circRNA interactions, TargetFinder was used to scan the miRNA-target sites of circRNA. The network of miRNAs–circRNAs was generated using Cytoscape 3.5.1 software. [...] Treated RNA solutions (10 μL, without DNA contamination) from seven stages, leaves, and roots were subjected to reverse transcriptase reactions with the PrimeScript RT Reagent Kit (Takara, Dalian, China), in accordance with the manufacturer’s protocol. Genomic DNA of A. thaliana was extracted using the Plant DNA Isolation Reagent (Takara, Dalian, China). To validate circRNAs identified in A. thaliana, polymerase chain reactions (PCRs) were performed using a set of divergent primers and a set of convergent primers that were used as a control (Supplementary Table ). The divergent primers were designed using an “out-facing” strategy to guarantee that the amplifications were from a circular template (). For each PCR amplification, 20 ng of cDNA or genomic DNA was used with rTaq DNA polymerase and 10× buffer (Takara, Dalian, China), and 35 cycles of PCR were performed. To confirm the PCR results, the PCR products were dissected from a gel and purified using MiniBEST Agarose Gel DNA Extraction Kit Ver. 4.0 (Takara, Dalian, China). Sanger sequencing was performed by Sangon Biotech Company (Shanghai, China).A qRT-PCR experiment was performed to test the expression levels of circRNAs. The qRT-PCR reactions contained 1 μL of diluted cDNA, 400 nM of each primer, 10 μL of the 2× TransStart Tip Green qPCR SuperMix, 0.4 μL of the 50× passive reference dye (TransGen Biotech, Beijing, China), and 7.8 μL of ddH2O, for a final volume of 20 μL. The following qRT-PCR program was used: denaturation at 94°C for 30 s, followed by 40 cycles of 94°C for 5 s, 55.5°C for 30 s, and 72°C for 10 s. The divergent primers used in the experiment are shown in Supplementary Table . Amplification results were analyzed using the comparative Ct method, which uses the formula 2-ΔΔCT. Each qRT-PCR experiment was carried out in three independent biological replicates and the standard errors of the mean among the replicates were calculated. Statistical analysis was conducted using the SPSS Statistics 18.0 software (IBM Corporation, Armonk, NY, United States). The significance of differences between leaves and roots was analyzed using a t-test at the probability level of 0.05. […]

Pipeline specifications

Software tools NGS QC Toolkit, TopHat, Bowtie2, find_circ, BEDTools, BLASTN, Blast2GO, Targetfinder, SPSS
Databases TAIR PlantcircBase
Applications Miscellaneous, RNA-seq analysis
Organisms Arabidopsis thaliana, Homo sapiens