library_construction_protocol: To characterize the cell subtype population of spinal cord motor neuron organoid induced in DNSCM and DASCM, single-cell RNA sequencing was performed using 10x Genomics. To digest the spinal cord motor neuron organoids (day28) into single cells, the DNSCM encasing the organoids was first removed by incubating with 2mg/mL collagenase Ⅰ at 37℃ for 15min. Organoid spheroids were collected by centrifugation at 500g for 5min and incubated with Accutase at 37 ° C for 5min. After digestion, the cell suspension was formed by gently blowing up and down with 1mL tips. The dissociated cells were filtered using a 40um cell strainer and then centrifuged at 500g for 5min, resuspended in the neurobasal medium. After measuring the density and viability of scMN-Organ-dissociated cells, the final cell density was modified into 700-1000 cells/μL. Dissociated cells were loaded onto the Chromium single cell controller (10x Genomics) to generate single cell gel beads in the emulsion according to the manufacture's protocol. Captured cells were lysed and the released RNA were barcoded through reverse transcription in individual GEMs. Reverse transcription was performed on a S1000TM Touch Thermal Cycler (Bio Rad) at 53°C for 45 min, followed by 85°Cfor 5 min, and hold at 4°C. The cDNA was generated and then amplified, and quality assessed using an Agilent 4200. Single-cell RNA-seq libraries were constructed using Single Cell 3' Library and Gel Bead Kit V3.1 The libraries were finally sequenced using an IlluminaNovaseq6000 sequencer with a sequencing depth of at least 100,000 reads per cell with pair-end 150 bp (PE150).