Cfx96 c1000 touch real time detection system

Young and aged killifish were euthanized in 0,1% buffered tricaine and the telencephalon was extracted and quickly snap-frozen on dry ice. The RNeasy Lipid Tissue Mini Kit (74804, Qiagen) was used to extract RNA, following the Manusfacturer’s instructions. cDNA was generated using oligo dT primers and Superscript III reverse transcriptase (Invitrogen). RT qPCR was performed using SYBR Green master mix (BioRad) and the CFX96/C1000 Touch Real-Time detection system (BioRad) with an annealing temperature of 60 °C. Plate set up was performed in CFX Maestro (BioRad v2.3). Reactions were run in duplo. Primer sequences for genes of interest and housekeeping genes (Supplementary Table 4) were designed based on the N. furzeri Transcriptome browser (www.NFIN.com). Housekeeping genes (Supplementary Table 4) were used to normalize the gene expression values. Values are quantified using the comparative Ct method, with the mean of the young/vehicle-treated cycle threshold values as the control.

Young and aged killifish were euthanized in 0.1% buffered tricaine, and brains were extracted and placed on a sterile petridish. Using a 25 Gauge needle, the region surrounding the injury site was quickly punched‐out, based on DiD positivity, to ascertain that our investigations were restricted to the injury area. Tissue was rapidly snap‐frozen on dry ice, and tissue from two fish was pooled to increase sample size. RNA was extracted using the RNeasy Lipid Tissue Mini Kit (74804, Qiagen) following the manufacturer instructions. RNA was converted into cDNA using oligo dT primers and Superscript III reverse transcriptase (Invitrogen). All qPCR reactions were run in duplo with an annealing temperature of 60°C, using SYBR Green master mix (BioRad) and the CFX96/C1000 Touch Real‐Time detection system (BioRad). Gene expression values were normalized against reference genes (qBase software, Biogazelle, Table S4). Values are quantified using the comparative Ct method, with the mean of the young cycle threshold values as the control. Primer sequences (Table S4) were designed based on the N. furzeri Transcriptome browser, available on NFIN (www.NFIN.com).