Specific reverse transcription kit

RNA isolation was performed using Trizol reagent (Solarbio, Beijing, China). Then, the complementary DNA (cDNA) was synthesized using the specific reverse transcription kit (Vazyme, Nanjing, China). Subsequently, the RNA level was examined via AceQ qPCR SYBR Green Master Mix (Vazyme) and quantified via the 2^−ΔΔCt method. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) acted as the internal reference to normalize the expression of circ_0005615 and NOTCH1, and U6 was used as the endogenous control to normalize the expression of miR-665. All primers were shown: circ_0005615-F: 5′-TCACCCTTTACCTGGAGCAAA-3′, circ_0005615-R: 5′-GAGCTGAAACGATGGTGACAAA-3′; miR-665-F: 5′-ACCAGGAGGCTGAGGC-3′, miR-665-R: 5′-GAACATGTCTGCGTATCTC-3′; NOTCH1-F: 5′-GAGGCGTGGCAGACTATGC-3′, NOTCH1-R: 5′-CTTGTACTCCGTCAGCGTGA-3′; GAPDH-F: 5′-GAAGGTGAAGGTCGGAGT-3′, GAPDH-R: 5′-GATGGCAACAATATCCACTT-3′; and U6-F: 5′-CTCGCTTCGGCAGCACA-3′, U6-R: 5′-ACGCTTCACGAATTTGCGT-3′.

TRIzol reagent (Leagene, Beijing, China) was applied for extracting total RNA. Afterwards, cDNA was synthesized using the specific reverse transcription kit (Vazyme, Nanjing, China). For detecting RNA levels, qRT-PCR reactions were carried out using SYBR Green Master Mix (Vazyme). RNA levels were quantified via the 2^−ΔΔCt method. GAPDH (for circ_0003204 and HDAC9) and U6 (for miR-942-5p) were regarded endogenous controls. The primers included: circ_0003204-F: 5′-C A T G G G G C T G T G T C A C C T G-3′, circ_0003204-R: 5′-G G C A A C T G G T G T G G A A G A G A-3′; miR-942-5p-F: 5′-C T T C T C T G T T T T G G C C A T G T G-3′, miR-942-5p-R: 5′-C T C T A C A G C T A T A T T G C C A G C C A C-3′; HDAC9-F: 5′-A G T A G A G A G G C A T C G C A G A G A-3′, HDAC9-R: 5′-G G A G T G T C T T T C G T T G C T G A T-3′; GAPDH-F: 5′-G C T G A G T A C G T C G T G G A G T C-3′, GAPDH-R: 5′-A G T T G G T G G T G C A G G A G G C-3′; U6-F: 5′-C T C G C T T C G G C A G C A C A-3′, U6-R: 5′-A A C G C T T C A C G A A T T T G C G T-3′.