GSM7770394: Organoid cells, scram shRNA, scRNAseq; Homo sapiens; RNA-Seq
At week 8, organoids were processed for single-cell transcriptomics using the PIPseq™ T2 3' Single Cell RNA Kit v4.0 (Fluent Biosciences). Cortical organoids were dissociated into single-cell suspensions via 30-min incubation at 37 ºC in papain and DNAse in EBSS (Cat. # LK003150, Worthington), shaking vigorously every 5-10 min. Samples were then triturated with manual pipetting 10 times, and pelleted with a 5 min spin at 300 g in 4 ºC. For hESC 1 (UCLA 6) samples, pellets were resuspended in the provided Cell Resuspension Buffer (Fluent Biosciences), and 5,000 cells were targeted for capture. For hESC 2 (UCLA 1) samples, pellets were resuspended in 2% FBS in PBS, then passed through a wet 35 µm cell strainer into round-bottom tubes for flow cytometry assisted cell sorting (FACS). Samples were treated with DAPI (1:1000) to identify dead cells, and processed on a FACSAria (BD Biosciences). FACS gates were set using uninfected cells, and mCherry– and mCherry+ sorted samples were pelleted once more and resuspended in 5 µL of 2% FBS in PBS. This process enabled the targeting of ~ 25,000 cells for capture, with the exception of mCherry+ cells from FEZF2 #1-shRNA-transduced organoids (7,500 cells targeted). Sequencing libraries were obtained from cells according to PIPseq T2 instructions, using 16 – 17 cycles for cDNA amplification and 8 – 12 cycles for library amplification depending on the amount of DNA. Libraries were quantified on an Agilent 2100 Bioanalyzer, and and sequenced as per manufacturer recommendation on a NovaSeq S2 flow cell.