title: GSM5915218: Cochlea E15.5 PVcre;Ai14 [SS2_20_151]; Mus musculus; RNA-Seq
library_construction_protocol: The same dissociation protocol was used for SGs of all stages. First, the presence of tdTomato signal was verified under a fluorescence stereomicroscope. From tdTomato positive animals, the cochlea was carefully dissected from the temporal bone as described above. Upon removal of the cochlear capsule, the spiral ganglia (SG) were dissected and collected on ice in Leibovit'z L-15 medium (Life technologies). For E14.5 and E15.5 tissue sample, the cochlea was cut open, and the modiolus region was dissected out. Tissue samples were then digested in a papain-DNAse solution (1.5 ml of papain at 1 mg/ml, 0.5 ml of DNAse at 0.1%) for 20 min, shaking at 700 RPM. After centrifugation of the partially digested tissue at 400 RCF for 10 min, the dissociation mix-solution was removed, and the cell pellet gently resuspended in Dulbecco's modified Eagle's medium (DMEM) F-12 (Life technologies). Subsequently, to break up the tissue fragments, cells were mechanically triturated using fire polished Pasteur pipettes coated with 0.2% bovine serum albumin until a homogenized solution was achieved. To remove residual cell aggregates and to generate a single cell suspension, the cell homogenate was then passed through a 70μm nylon cell strainer (BD Biosciences). Single RFP+ cells were sorted by fluorescence-activated cell sorting (FACS) into individual wells containing lysis buffer in a 384-well plate. The plates were immediately placed on dry ice and stored at -80°C before processed for Smart-seq2 protocol. We used 3-4 animals (6-8 cochlea) per experiment, with several rounds of experiments and plates per time-point. RNA libraries were prepared according to Smart-seq2 protocol.