GSM9016771: Skin, WT rep1; Mus musculus; RNA-Seq
Animals were euthanized and ears were collected and digested with 3 mg/mL Collagenase D (Roche) and 10mM DNase I (Roche) in serum-free medium RPMI for 60 min at 37℃. Enzyme activity was inhibited by adding 10 mL of PBS supplemented with 1% of FBS. The tissues were then mechanically disrupted and filtered through a 100 mm cell strainer to obtain a single cell suspension. For surface staining, cells were stained with antibodies against surface antigens in FACS buffer (PBS supplemented with 1% FBS) on ice for 30 minutes using appropriate antibodies. We employed the 10x Genomics Chromium Single-Cell 5' v2 kit for library preparation, strictly adhering to the manufacturer's guidelines. Breifly, CD45+ cell suspensions were loaded onto the 10x Genomics Chromium Controller, where individual cells were partitioned into gel dead-in emulsions (GEMs) along with barcoded beads. Within each GEM, cells were lysed and reverse transcription occurred, tagging each cDNA molecule with a cell-specific and molecular barcode (UMI). After GEMs were broken, the pooled cDNA was purified and amplified via PCR. Amplified cDNA was used to construct 5′ gene expression libraries. The resulting libraries were then quantified, quality-checked, and prepared for sequencing on an Illumina platform.