The 24 glia-secreted factors selected for screening experiments (Fig. 1D, S3D–3E) were produced from HEK293T cells transfected with plasmids encoding human proteins by a standard calcium phosphate protocol (Zhang et al., 2013). The culture medium was collected 72 hours after transfection, filtered with a 0.2 µm syringe filter to remove cell debris, diluted (1:5) with fresh growth medium Neurobasal-A/B27, and added into cultures of human neurons on MEFs at D10. At D12, the culture medium of neuronal cultures was harvested for Aβ ELISA (Fig. 1D), immunoblotting and qRT-PCR for APP and several control proteins (Fig. S3D–S3E). The human gene cDNA plasmids used for transfection were acquired by Gateway recombination cloning (Invitrogen). The destination vector is pLX304 (CMV promoter, Addgene plasmid # 25890), and the following clones were obtained from Harvard Medical School: APOE, HsCD00044600; CLU, HsCD00040309; CP, HsCD00348042; CST3, HsCD00043456; CTGF, HsCD00378376; CYR61, HsCD00040135; FN1, HsCD00296172; FST, HsCD00042431; IGF2, HsCD00366367; IGFBP2, HsCD00076522; IGFBP5, HsCD00368361; LGALS12, HsCD00369800; HsCD00323559; METRNL, HsCD00339900; NENF, HsCD00377723; NPVF, HsCD00295230; PTN, HsCD00082359; SERPINF1, HsCD00371068; SPARC, HsCD00042420; SPP1, HsCD00040432; THBS1, HsCD00437810; THBS2, HsCD00379263; TIMP1, HsCD00039987; WNT5A, HsCD00375640; WNT7A, HsCD00042006. The control plasmid is FUGW (EGFP driven by CMV promoter, Addgene plasmid # 14883)
