GSM8971817: CX3CR1-EGFP and NG2DsRed cells; Mus musculus; RNA-Seq
CX3CR1EGFP; NG2DsRed mice at P15 were used for scRNA-seq. Temporal bones were harvested from 10 mice (20 capsules) and placed in ice-cold RPMI medium. Eardrums were dissected and transferred to 1.5 mL microcentrifuge tubes containing 1 mL of RPMI medium supplemented with 1.5 mg/mL collagenase IV (Gibco, Cat# 17104019) and 1 mg/mL DNase I (Roche, Cat# 10104159001). Tissues were minced with sterile micro scissors and digested at 37°C for 30 minutes with gentle shaking (100 rpm Following digestion, the mixture was centrifuged at 300 × g for 5 minutes at 4°C. The pellet was further dissociated in TrypLE Express (Gibco, Cat# 12604013) at 37°C for 10 minutes, with pipette trituration every 5 minutes using a 1000 μL tip to generate a single-cell suspension. The suspension was passed through a 70 μm cell strainer (Falcon), washed once with PBS, and resuspended in 0.04% BSA in PBS with DAPI. Alive cells were sorted using FACS (Influx-Kirk) according to the presence or absence of EGFP and DsRed signal using a C57BL6 mouse as negative control. Purified cells were resupspended in 0.04% BSA in PBS. Libraries were prepared using the Chromium Next GEM Single Cell 3ʹ Reagent Kits v3.1 (10x Genomics), following the manufacturer's protocol. Briefly, 4,860 cells (87% viability; estimated recovery: 2,859 cells) were resuspended in the master mix and loaded onto a Chromium chip along with gel beads and partitioning oil to generate gel bead-in-emulsions (GEMs).  Within each GEM, cells were lysed and poly-A RNA transcripts were reverse transcribed into cDNA. Each cDNA molecule incorporated an Illumina R1 primer sequence, a Unique Molecular Identifier (UMI), and a 10x barcode during this step. Barcoded cDNA was then pooled, purified using Silane DynaBeads, and amplified by PCR. Size selection of the amplified products was performed using SPRIselect reagent. Approximately 50 ng of cDNA was obtained after 14 cycles of amplification, and 29 ng was used for library construction. During this process, the Illumina R2 primer sequence, paired-end adapters (P5 and P7), and a sample index were added.