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Fig. 2 | Genome Biology

Fig. 2

From: Single-cell RNA-seq transcriptome analysis of linear and circular RNAs in mouse preimplantation embryos

Fig. 2

Detection of circRNAs by SUPeR-seq in individual HEK293T cells and mouse preimplantation embryos. a Identification of circRNAs in the SUPeR-seq dataset. The termini of two exons are in a sequential order (a–-b……c–-d) along the genome, the red part shows the upstream exon at the 5′ end of the gene while the blue part shows the downstream exon at the 3′ end of the gene (top). When looped, the two exons join together from head to tail in a reversed order (cdab, bottom). Various sequencing reads covering the junction site can identify the cyclization of a circRNA. b Sanger sequencing validation of a newly discovered circRNA through SUPeR-seq in a single HEK293T cell. The end-joining region of the circRNA is PCR amplified to confirm the reversed order of the joined exons. The sequence of the end-joining region is unique to the circRNA but not the host linear RNA. The joint region of the circRNA is chr7:11021999–11030474, and the host gene is PHF14. c Quantitative RT-PCR of HEK293T cell total RNA treated with RNase R or mock treatment as a control. The circRNA candidates showed ten- to hundred-fold enrichment compared with common linear mRNAs after treatment with RNase R (here we use Gapdh, Rps24 and Actb as linear RNA controls). This clearly demonstrates that all the circRNA candidates are circular. For each circRNA, we made two replicates in the RT-qPCR step. d The length distribution of the circRNAs (141 in total) detected in single HEK293T cells. e The length distribution of circRNAs (2891 in total) detected in mouse preimplantation embryos. f The top GO terms are displayed for 1316 genes from which circRNAs are generated in mouse preimplantation embryos

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