RNA isolation and RT-qPCR

Total RNA from mouse neuroretinas and cell lysates was extracted using the RNeasy Mini Kit (QIAGEN, Hilden, Germany) and the same amount of RNA was transcribed into cDNA using the SuperScript II Reverse Transcriptase Kit (Thermo Fisher Scientific) following the manufacturer's instructions. Transcript levels of genes were analysed by RT-qPCR performed using a Roche LightCycler 480 (Roche, Basel, Switzerland) with either TaqMan assays or SYBR Green detection as previously described (Harkin et al., 2022; Augustine et al., 2018). Relative gene expression was calculated using the comparative Ct method (2^−ΔΔCt) with data normalised to Actb or Rn18s. Roche-validated mouse TaqMan assays were purchased for Ccl2 (310467), Il1b (310471), Il18 (301115), Il33 (316824), Il6 (300699), Tgfb (317139), Tnf (317484), Vegf (314944), Actb (307903) and Rn18s (307906). SYBR Green gene-specific primers were designed as previously described (Augustine et al., 2018) and the primer sequences are listed in Table S2.