title: GSM7595894: iCtrl, 30d, scRNA-seq; Homo sapiens; RNA-Seq
library_construction_protocol: Midbrain organoids were collected from their culture medium and washed with 1x PBS (phosphate-buffered saline, Gibco, cat. nr. 10010-23). Organoids were digested in 1 ml sCelLive™ Tissue Dissociation Solution (Singleron Biotechnologies, cat. nr. 1190062) in a 15 ml conical tube (Sarstedt, cat. nr. 62.5544.003) and placed in a thermal shaker at 50 rpm at 37°C for 30 minutes. The state of dissociation was checked at regular intervals under a light microscope. Following digestion, the suspension was filtered using a 40-µm sterile strainer (Greiner, cat nr. 542040). The cells were centrifuged at 350xg for 5 minutes at 4°C, and the cell pellets were resuspended in 500µl PBS. Cells were stained with Acridine Orange/Propidium Iodide Stain (Logos Biosystems, cat. nr. F23001), and the cell number and viability were calculated using LUNA-FX7™ Automated Cell Counter (Logos Biosystems, Villeneuve d'Ascq, France). The single-cell RNA-seq libraries were constructed using GEXSCOPE™ Single Cell RNAseq Library Kit (Singleron Biotechnologies, cat nr. 4161031) according to the manufacturer´s instructions. Briefly, for each library, the concentration of the single-cell suspension was adjusted to 3x105 cells/ml with PBS, and the suspension was loaded onto an SD microfluidic chip to capture 6000 cells. Paramagnetic beads conjugated to oligodT probes that carry a unique molecular identifier (UMI) and a barcode unique to each bead (from the same kit) were loaded, after which the cells were lysed. The beads bound to polyadenylated mRNA were extracted from the chip and reverse transcribed into cDNA at 42°C for 1.5 hours, and the cDNA was amplified by PCR. The cDNA was then fragmented and ligated to indexed Illumina adapters. The fragment size distribution of the final amplified library was obtained on an Agilent Fragment Analyzer.