Neuron Isolation from Co-Cultures

To remove neurons from cocultures, prewarmed PBS without Ca²⁺/Mg²⁺ (Thermo Fisher Scientific) was applied to the wells to wash the cells followed by a solution of 0.5 mM EDTA in PBS without Ca²⁺/Mg²⁺. Plates were incubated for a few minutes at 37°C and then observed under the microscope while gently rocking. Detached neurons were then removed, and wells were washed again with PBS without Ca²⁺/Mg²⁺. For RNA isolation, cells of 6 technical repeats were lysed in RNA isolation buffer containing 1% 2-mercaptoethanol (Thermo Fisher Scientific) and isolated with Quick-RNA MicroPrep according to the manufacturer’s protocol (Zymo Research). All samples were checked for RNA integrity number values greater than 9, and mRNA sequencing library was done according to protocol (Illumina). Libraries were sequenced on a NovaSeq S1 flowcell, SR100 mode, with 30 million reads per sample. Raw Fastq data were aligned against the human reference transcriptome hg38 using Salmon (71 (link)), and data analysis was performed with the R software package limma (72 (link)). For DEGs, only genes with 2-fold differences and below an adjusted P value of 0.01 were included. For pathway and Gene Ontology (GO) analysis, the web database Enrichr was used (70 (link)).