GSM7721027: OVI15M; Mus musculus; RNA-Seq
The fresh C57BL/6 murine ovaries, oviducts, and uteri were excised from each age group (5D, 5W, 3M, 6M, 9M, 12M, and 15M, n = 6/age group). Tissues from the same organ were then pooled together. Within the first 30 min of the procedure, the collected fresh tissues were placed on ice and soaked in sCelLiveTM Tissue Preservation Solution (Singleron Biotechnology, China). The specimens were cleaned thrice with Hanks Balanced Salt Solution and then digested by Singleron PythoNTM Automated Tissue Dissociation System (Singleron Biotechnology, China) using 3 mL sCelLiveTM Tissue Dissociation Solution for 15 min at 37 °C. Next, the cells were incubated at 25 °C for an additional 10 min to remove the red blood cells following the addition of GEXSCOPE® red blood cell lysis solution (2 mL, Singleron). The solution was then gently suspended in phosphate buffer saline (PBS, HyClone, China) after being centrifuged at 300 × g at 4 °C for 5 min to remove the supernatant. Finally, the samples were stained with trypan blue (Sigma, USA), and cell vitality was analyzed using an inverted microscope (Nikon, Japan). The Singleron Matrix® Single Cell Processing System was used to load single-cell suspensions (1 × 105 cells/mL) in PBS (HyClone) into the GEXSCOPE® Single Cell Microchip (Singleron Biotechnology, China). The scRNA-seq libraries were constructed according to the protocol of the GEXSCOPE® Single Cell RNA Library kits (Singleron). Briefly, following the removal of the barcoding beads from the microwell chip, reverse transcription was performed on the mRNA that attached to the beads to produce cDNA, and PCR amplification was then performed. Following fragmentation, the amplified cDNA was ligated using sequencing adapters. GEXSCOPE® Single Cell RNA Library Kits (Singleron) were used for the construction of scRNA-seq libraries, in accordance with the manufacturer's instructions. On an Illumina NovaSeq 6000 with 150-bp paired-end reads, individual libraries were combined after being diluted to 4 nM.