Cyanine3 (cy3)

FISH assay was performed following the experiment protocols in previous report [17 (link)]. PBS was used to wash HCT116 and SW480 cells (5 × 10⁴) grown on the slides, followed by 30-min fixation with 4% paraformaldehyde. Then, the slides were treated with DNase to digest potentially contaminating DNA at 37 °C. Subsequent to incubation in prewarmed hybridization buffer at 55 °C for 2 h, the LINC00665 FISH probe or miR-138-5p FISH probe tagged by Cy3 (Ribobio) was added onto the culture dishes for hybridization at 65 °C overnight. After fostering with RNase A and blocking solution, the culture dishes were washed and treated with DAPI solution for staining. Finally, the stained cells were analyzed by fluorescent microscope. Sequences of LINC00665 biotin probe and miR-138-5p biotin probe were listed in Additional file 7: Table S4.

The probe against LINCO2820 was marked with cy3, which was designed by RiboBio (Guangzhou, China). The 2 × 10⁴ cell samples were placed in 8-well glass slides (Millipore) until they adhered to the slides. All subsequent steps followed the manufacturer’s directions. Finally, confocal microscopy was used to obtain the images.

A direct method for FISH was implemented based on the manufacturer's instructions to detect circPAPPA2 in TEV1 cells. Briefly, a circPAPPA2 probe was synthesized and labelled with the fluorescent substance Cy3 (RiboBio, Guangzhou, China). After fixation with 4% paraformaldehyde and treatment with 0.5% Triton X-100, cells were cultured with hybridization solution containing no probe for prehybridization to block nonspecificity. The hybridization reaction was carried out with a hybridizing solution containing a labelled probe (sequence provided in Supplementary Table S3) and cells overnight at 37 °C. Cellular DNA was counterstained with 4ʹ,6-diamidino-2-phenylindole (DAPI).