GSM9266939: Zebrafish brain tissue cells, control group; Danio rerio; RNA-Seq
Fresh tissues were placed in sCelLive™ tissue preservation solution (Singleron) on ice within 30 minutes after surgery. The specimens were washed three times with Hanks Balanced Salt Solution (HBSS), minced into small pieces, and then incubated with 3 mL of sCelLive™ tissue dissociation solution (Singleron) using the Singleron PythoN™ tissue dissociation system at 37°C for 15 minutes. The cell suspension was collected and filtered through a 40 μm sterile filter. Subsequently, GEXSCOPE® Red Blood Cell Lysis Buffer (RCLB, Singleron Matrix™) was added, and the mixture was incubated at room temperature for 5–8 minutes (following the abovementioned ratio) to remove red blood cells. Afterward, the mixture was centrifuged at 4°C and 300 × g for 5 minutes. The supernatant was discarded, and the pellet was gently resuspended in PBS. Finally, the sample was stained with trypan blue, and cell viability was assessed under a microscope. The single-cell suspension (2×10⁵ cells/mL, diluted with HyClone PBS) was loaded onto the Singleron Matrix® single-cell processing system's microfluidic chip. Subsequently, barcoded magnetic beads were collected from the microfluidic chip. mRNA captured by the barcoded magnetic beads was reverse-transcribed to generate cDNA, followed by PCR amplification. The amplified cDNA underwent fragmentation and was ligated to sequencing adapters. The scRNA-seq library was constructed using the GEXSCOPE® single-cell RNA library kit (Singleron) according to the manufacturer's protocol. The libraries were diluted to 4 nM, pooled, and sequenced on the Illumina NovaSeq 6000 platform with 150 bp paired-end reads.