GSM8365314: young mice; Mus musculus; RNA-Seq
2-month-old and 24-month-old mice were anesthetized with Avertin (250 mg/kg) by i.p. injection. After dissection, the testes were immediately placed in ice-cold saline. After fully washing the testes with PBS, the tunica albuginea was carefully removed. The testes were mechanically minced into pieces using microscopic forceps and digested with accutase (Invitrogen, USA) at 37°C for 5 minutes. The accutase activity was halted by adding DMEM/F12 supplemented with 10% fetal bovine serum (Gibco, USA), and the samples were rinsed twice with PBS. The obtained cell suspension was filtered through a 45 μm strainer and centrifuged. At last, cells were resuspended in phosphate-buffered saline (PBS) containing 0.1% BSA (Thermo Fisher Scientific, USA) at a concentration of ~1,000 cells/μL for single-cell sequencing. The BD Rhapsody system was used to capture the transcriptomic information. Single-cell capture was achieved by randomly distributing a single-cell suspension across microwells using a limited dilution approach. Beads with oligonucleotide barcodes were added in excess to ensure that a bead paired with a cell in each microwell. Cell-lysis buffer was added to allow poly-adenylated RNA molecules to hybridize to the beads. The beads were collected into a single tube for reverse transcription. Whole transcriptome libraries were prepared using the BD Rhapsody single-cell whole-transcriptome amplification workflow. Sequencing libraries were quantified using a High Sensitivity DNA chip (Agilent, USA) on a Bioanalyzer 2200 and the Qubit High Sensitivity DNA assay (Thermo Fisher Scientific, USA). The library for each sample was sequenced using Illumina sequencer (Illumina, CA).