Savant sdp121 p

Cerebral organoids (COs) were washed with PBS, treated for 1 h with cell recovery solution (CRS, Corning, NY, USA) at 4 °C, and washed with PBS again. CO was homogenized in a microtube with a glass bead (Benchmark Scientific, Edison, NJ, USA). The COs were stored at -80 °C before processing. COs were freeze-dried (Savant SDP121 P, SpeedVac, ThermoFisher Scientific, USA) and homogenized using a glass bead. Lipid, ganglioside, and metabolite extraction were performed by adding 100 µl of 80% isopropanol to the homogenate. It was followed by a brief vortex, sonication (37 Hz, 5 min), and vortexing (200 rpm, 10 min). The extract was then centrifuged (12.3 RCF, 5 min), and the filtrate was removed and mixed 1:1 with a mixture of lipid and metabolite internal standards (Supplementary Table 2) for lipidomics, and metabolite assays or with a mixture of isotopically labeled GM1 and GM3 internal standards for ganglioside assay, and stored in -20 °C until LC-MS analysis. The protein pellet was dried using the SpeedVac vacuum concentrator (Savant SDP121 P, ThermoFisher Scientific). The dried protein pellet was reconstituted to perform the BCA assay protein determination. Control (N = 10) and tramiprosate-treated COs (N = 10) were utilized for the selective reaction monitoring - mass spectrometry (SRM-MS) analysis. The protein pellet was dried to be processed for the proteomics assay.