GSM7732412: Striatum organoids; Homo sapiens; RNA-Seq
Single nuclei suspension was obtained using the Minute Single Nucleus Isolation Kit for Neuronal tissues/cells (Invent Biotechnologies, INC.). To homogenize the frozen organoids, a pellet was first formed and then placed in an Eppendorf tube. 200μl of cold buffer A were added, and the mixture was pipetted up and down with a P200 pipette for 30 seconds. After that, 500μl of Buffer A were added, and the suspension was incubated on ice for 20 minutes. Every 5 minutes during incubation, pipetting up and down with a P200 for 30 seconds was performed to fully dissociate the organoids. The level of dissociation was observed under a light microscope at 5-minute intervals. After homogenization and dissociation, the suspension was transferred to a filter in a collection tube and then directly centrifuged at 13,000 X g for 30 seconds. The filter was discarded, and the resulting pellet was gently resuspended by pipetting up and down for 10-20 times and then centrifuged at 600xg for 5 minutes. The pellet containing nuclei was then resuspended in 500 μl of cold nuclei suspension buffer. Trypan Blue staining (0.4% w/v, Gibco) staining was used to assess the quality of the nuclei under a light microscope, propidium iodine with a Luna FX7 automated cell counter (Logos Biosystems, Villeneuve d'Ascq, France) was used to count the nuclei. The reverse transcription to cDNA and the library were prepared with the GEXSCOPE® Single Nucleus RNA Library Kit V2 (Singleron Biotechnologies). A total of 30,000 nuclei were utilized and loaded onto a microfluidic chip (Singleron GEXSCOPE® Single Nucleus RNA Library Kit V2) to achieve a minimum capture of 6,000 nuclei. In a brief overview of the process, barcoded beads containing unique Cell Barcodes were loaded onto the chip, and then the nuclei were lysed. Following the nuclei lysis, Barcode Beads using poly (dT) sequences captured the polyadenylated RNA. The Barcode Beads, now containing captured mRNA molecules, were collected and subjected to a reverse transcription reaction. Subsequently, the cDNA was amplified and subjected to quality control (QC) procedures.