GSM9340800: Tie2-GFP cells; Mus musculus; RNA-Seq
Temporal bones were harvested from 1–3 month-old Tie2-GFP or NG2-DsRed reporter mice (n = 10 per batch; 20 cochleae) immediately after euthanasia and placed in ice-cold Leibovitz's L-15 medium (Gibco, Cat# 11415064). Under a dissecting microscope, the cochlear lateral wall was opened from apex to base, and the stria vascularis (SV) was carefully microdissected from each cochlea. For each mouse line, SV tissue from both ears was pooled in 1.5 mL microcentrifuge tubes containing L-15 on ice. Pooled SV tissue was transferred to 1 mL of L-15 supplemented with collagenase IV (Gibco, Cat# 17104019) and DNase I (Roche, Cat# 10104159001), minced with sterile fine scissors, and digested at 37°C for 30 minutes with gentle shaking (~100 rpm). After collagenase digestion, the enzyme solution was removed and replaced with an EBSS-based papain solution (Gibco, Cat# 14170161) containing papain (20 U/mL; Worthington Biomedical, Cat# LS003118), 1 mM L-cysteine (Sigma Aldrich, Cat# C7352), 0.5 mM EDTA (Invitrogen, Cat# 15575020), 15 mM HEPES (Gibco, Cat# 15630080), and DNase I. Samples were incubated for an additional 30 minutes at 37°C, and the tissue was gently triturated every 10 minutes using a 1000 μL pipette tip to generate a single-cell suspension while minimizing mechanical stress. Papain activity was quenched by adding an equal volume of Leibovitz's medium containing 20% ovomucoid protease inhibitor (Worthington, Cat# LS003085). The suspension was passed through a 20–70 μm cell strainer to remove undigested fragments, and flow-through cells were collected and centrifuged at 300 × g for 5 minutes at 4°C. The cell pellet was washed twice with PBS containing 0.04% BSA and resuspended in 0.04% BSA/PBS. An aliquot of each suspension was mixed with trypan blue (Gibco, Cat# 15250061) and counted using an automated cell counter; only preparations with >80–85% viable cells were used. For enrichment of vascular populations, alive cells were sorted by FACS according to Tie2-GFP or NG2-DsRed fluorescence, using C57BL/6 tissue as a negative control and forward/side scatter to exclude debris and doublets. Purified cells were collected into tubes containing 0.04% BSA in PBS on ice, counted, and resuspended in 0.04% BSA/PBS at the appropriate concentration for loading onto the 10x Genomics Chromium system. Libraries were prepared using the Chromium Next GEM Single Cell 3ʹ Reagent Kits v3.1 (10x Genomics), following the manufacturer's protocol. Briefly, 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.