GSM8586698: DMD; Homo sapiens; RNA-Seq
The single nuclei suspension was obtained using the GEXSCOPE® Single Nucleus RNA Library Kit V2 (Singleron Biotechnologies) following manufacturer' instructions. Briefly, EHT tissues were placed in cold nucleus separation solution on ice and cut into small pieces using ophthalmic scissors. Further homogenization was achieved by performing 5 strokes with pestle A and 5 strokes with pestle B of the Kimble douncer (KIMBLE® KONTES® Dounce Tissue Grinder). The sample was incubated on ice for 15 minutes where the state of dissociation was monitored every 5 minutes under a light microscope. Following homogenization and digestion, the suspension was filtered using a 40-μm sterile strainer (Greiner). The nuclei suspension was centrifuged at 200 × g at 4°C for 2 minutes, after which the supernatant was centrifuged at 500 × g for 5 minutes at 4°C to collect the nuclei. The resulting pellet was gently resuspended in 250 μL of cold nuclei suspension buffer. The quality of the nuclei was assessed by Trypan Blue staining (0.4% w/v, Gibco) under a light microscope. The nuclei were counted using propidium iodine with a Luna FX7 automated cell counter (Logos Biosystems). A total of 45,000 nuclei were loaded onto a microfluidic chip using the Singleron GEXSCOPE® Single Nucleus RNA Library Kit V2, targeting the capture of approximately 10,000 nuclei. Barcoded beads, each containing a unique cell barcode, were loaded into the chip alongside the nuclei, which were subsequently lysed to release cellular RNA. Following lysis, polyadenylated RNA molecules were captured by the poly(dT) sequence on the barcoded beads. The beads containing captured mRNA were then collected for reverse transcription to generate cDNA. The cDNA was amplified, followed by quality control (QC) checks. Illumina-compatible next-generation sequencing (NGS) libraries were generated and sequenced at Macrogen Europe BV facility (Amsterdam, The Netherlands) using a paired-end 150 bp read length Illumina™ Novaseq X Plus platform.