Gene Expression Analysis of Neural Markers

Gene expression analyses of selected markers were performed as described previously (Červenka et al., 2021 (link)). Briefly, total RNA was isolated from BG-differentiating NSCs and astrocyte conditions by RNeasy Plus Mini Kit (Qiagen) with QIAshredder (Qiagen), and converted into cDNA with QuantiTect Reverse Transcription Kit (Qiagen) according to the manufacturer's instructions. The reaction mix for one quantitative PCR contained 5× HOT FIREPol EvaGreen qPCR Mix Plus (Solis BioDyne), 125 nM of each primer (Table S4), 25 ng of cDNA template, and PCR water. Following settings were used on CFX96 Touch Real-Time detection system (Bio-Rad): 12 min at 95°C for enzyme activation, then 15 s at 95°C for denaturation with 40 cycles of 30 s at 57°C for annealing, and 30 s at 72°C for an extension. Cycle threshold (Ct) values were normalized to the average of two housekeeping genes Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and ATP synthase subunit beta, mitochondrial (ATP5F1B).