Quantifying Zebrafish Jagged Expression

All zebrafish experiments were carried out on the AB line zebrafish. All procedures were conducted in full accordance with the institutional guidelines approved by the Animal care and Use Committee of Xinhua Hospital (XHEC-WSJSW-2018-029). Total RNA was extracted from pools of 25 zebrafish embryos at different developmental time points using TRIzol reagent (Invitrogen, Cat # 9109, United States). The Primescript RT Master Kit (Takara, Catalyst # RR036A, Japan) was used to synthesize cDNA. To quantify the relative expression of jag1a and jag1b, q-PCR was performed using SYBR Green Master Mix (Applied Biosystems, Catalyst # A25742, United States) with fluorescent tags located on QuantStudio Dx Real Time PCR Instrument (Applied Biosystems, CA, United States). The primers for q-PCR were listed in Supplementary Table S4. The 18s ribosomal RNA (18-s) gene was used for standardization (McCurley and Callard, 2008 (link)). The relative expression levels of each sample with three independent triplicates were calculated using the RQ formula (RQ = 2^−ΔΔCT). To demonstrate the effectiveness of the splice-blocking morpholinos (SBMOs), RT-PCR was performed using Premix Taq™ (Takara, Cat#RR902A, Japan). The primers for RT-PCR were listed in Supplementary Table S5.