Fig. 6

In situ hybridization (ISH) and quantitative PCR validation of circRNA in pig embryonic brain. a Localization of circHDAC2 at E80. The circular isoform was detected by in situ hybridization using a 20-nucleotide alkaline-phosphatase conjugated LNA probe matching the back-splice junction (10 nucleotides on each side). Standard controls were conducted applying either RNase A or 100-fold excess of unlabeled probe. b, c Distribution of circHDAC2 in cortex. Detection of circHDAC2 or GAPDH (linear control) as described in (a) at E60 and E80, respectively. Treatment with proteinase K followed by RNase R reduced the GAPDH signal while not affecting circHDAC2, supporting the circular nature of the probe target. Proteinase K alone did not reduce either signal. Roman numerals indicate cortical layers. d, e Change in circRNA levels between E60 and E80. E60/E80 ratios for six different circRNAs, according to Illumina next generation sequencing (NGS) data (one sample) (d) and quantitative PCR (qPCR) on tissue used for ISH (two biological replicates, each done in triplicate) (e). f, g Subcellular localization of circHDAC2 (f) or circZEB1 (g) in the subplate at E60 and in layer IV/V at E80. Subcellular localization was visualized using panomics probes for high-resolution ISH and DAPI for nuclear localization. CP cortical plate, MZ marginal zone, SP subplate. Scale bars: 200 μm (a), 50 μm (b, c), and 10 μm (f, g)