Premature neural differentiation in cerebral organoids derived from early-onset autism individuals in a birth cohort

Diversity clinical phenotypes of autism spectrum disorders (ASD), caused by heterogeneity of genetic and environmental factors, hampered the investigation in uncovering pathological mechanisms. Here we generated ASD patient-cerebral organoids from induced pluripotent stem cells (iPSCs) that reprogramed from the early-onset ASD individuals at around 2 years old with common clinical diagnosis in a prospective birth cohort, which was excluded typical autistic mutations and environmental exposures. The organoids were dissociated by trypLE (Life technology) into single cells. The cell debris and other aggregates were removed by an additional low-speed centrifugation. About 12,000 cells were captured 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 degrees Celsius for 45 min, followed by 85 degrees Celsius for 5 min and final hold at 4 degrees Celsius. cDNA was generated and then amplified, and quality was assessed using an Agilent2100. scRNA-seq libraries were prepared with the Chromium Single Cell 3′ Library & Gel Bead Kit V3 (10x Genomics, 1000075) and then sequenced on an Illumina NovaSeq6000 with a sequencing depth of at least 50000 reads per cell with a paired-end 150 bp (PE150) reading strategy.