GSM1463873: E16Un; Mus musculus; RNA-Seq
Utricles were dissected from the temporal bone and (at postnatal stages) incubated for 2 minutes in protease XXIV (0.1 mg/ml) to remove the otoconia. Cochleas were dissected and freed from the spiral ganglion and Reisner’s membrane to expose the sensory epithelium. The organs were collected in DMEM (LifeTechnologies, )+5% fetal bovine serum (FBS) on ice. The cells were dissociated by incubating the organs in 1 mg/ml dispase (Gibco) and 1 mg/ml collagenase I (Worthington) in 100 µl for about 10-12 utricles or 200 µl for 10 to 12 cochleas for 30 to 45 minutes (depending on the developmental stage) at 37°C and triturating with a pipette. The dissociation was controlled visually with an inverted microscope. Dissociation buffer (Gibco 13151-014)-5% FBS was added to complete the dissociation and the samples then placed on ice. The dissociated tissues were filtered through a 40 µm cell strainer to eliminate clumps prior to sorting. Cells were sorted on a BD FACS Aria II cell sorter using a 100 µm nozzle and low pressure. Hair cells were collected using the brightest GFP signal and other cell types were collected using the lowest fluorescence signal. Cells were collected directly into a lysis buffer from the RNeasy Plus Micro kit (Qiagen). Total RNA extraction was performed using the manufacturer's recommendation and the quality was assessed with Agilent 2100 Bioanalyzer picochips. mRNA was amplified using the Ovation® 3'-DGE System (Nugen). 3’-enriched transcriptome tags were converted into non-directional Illumina sequencing libraries.