Sparkscript rt plus kit

Total RNA was extracted from 35 zebrafish larvae and murine neutrophil cells using a SPARKeasy Tissue/Cell RNA Rapid Extraction Kit (SPARKjade, AC0202, Shandong, China), and reverse transcription was performed using a SPARKScript RT plus Kit (SPARKjade, Shandong, AG0304). Real-time quantitative PCR (Q-PCR) was conducted using a SYBR Green kit (RR820A, Takara, Japan). Approximately 40 cycles of 95 °C for 10 s and 60 °C for 30 s were used to amplify the related genes, including tnf-α, il-1, il-6, cxcl8a, duox, sod1, cat, pkma, hdac3, and gapdh (Supplementary Table S1). The Q-PCR experiments were repeated with three individual biological samples. The data were normalized to the expression level of the housekeeping gene β-actin, and the relative expression levels were calculated using the 2^(−ΔΔCt) method.

Total RNA was extracted from cell aggregates using SPARKeasy Tissue/Cell RNA Rapid Extraction Kit (SPARKjade, AC0202, Shandong, China) and reverse transcription was performed using a SPARKScript RT plus Kit (SPARKjade, AG0304). A Light Cycler SYBR Green I Master (Roche, 04707516001, Mannheim, Germany) kit was used to prepare PCR mix and PCR was performed using a Light Cycler 480 Real-Time PCR System (Roche, Mannheim, Germany). The relative gene expression was calculated using the 2^{−(△△Ct)} method and Gaphd was used as a housekeeping gene. Primer sequences used are listed in Additional file 2: Table S2.