GSM5034746: Cochlea P3 PVcre;Ai14 [SS2_15_0135]; Mus musculus; RNA-Seq
The same dissociation protocol was used for SGs of E16.5, E18.5 and P3 PVCre;Ai14 mice. First, the presence of tdTomato signal was verified under a fluorescence stereomicroscope. From tdTomato positive E16.5, E18.5 and P3, the cochlea was carefully dissected from the temporal bone as described above. Upon removal of the cochlear capsule, the spiral ganglia (SG) and HCs were dissected and collected on ice in Leibovit'z L-15 medium (Life technologies). The SG 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 (embryonic samples) or 35 min (for P3 samples), 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 7 animals (14 cochlea) per time-point. RNA libraries were prepared according to Smart-seq2 protocol.
GEO Accession
GSM5034746