GSM9060373: P7_Ctrl_IHC_1; Mus musculus; RNA-Seq
Cochlear samples were incubated in choline chloride solution containing 20 U/mL papain (Cat# LK003178, Worthington) and 100 U/mL DNase I (Cat# LK003172, Worthington) for 20 min at 37°C, and this was followed by digestion with a protease (Cat# P5147, Sigma; 1 mg/mL) and dispase (Cat# LS02104, Worthington; 1 mg/mL) for 20 min at 25°C. The components of the choline chloride solution contained 92 mM choline chloride, 2.5 mM KCl, 1.2 mM NaH2PO4, 30 mM NaHCO3, 20 mM HEPES, 25 mM glucose, 5 mM sodium ascorbate, 2 mM thiourea, 3 mM sodium pyruvate, 10 mM MgSO4.7H2O, 0.5 mM CaCl2.2H2O, and 12 mM N-acetyl-L-cysteine. Next, the post-digested cochlear samples were gently triturated using fire-polished glass Pasteur pipettes (13-678-20b; Fisher) with 4 different pore sizes (largest size the first, and smallest one the last). The picked endogenous IHCs and iOHCs were immediately subjected to reverse-transcription and cDNA amplification using a Smart-Seq HT kit (Cat# 634437, Takara). Smart-Seq HT kit (Cat# 634437, Takara) was used to generate full-length cDNA. The cDNAs (1 ng each) were tagmented using a TruePrep DNA Library Prep Kit V2 for Illumina (Cat# TD503-02, Vazyme) and a TruePrep Index Kit V2 for Illumina (Cat# TD202, Vazyme).