Time-series transcriptome analysis of the older adult mouse cochlea after noise exposure
Time-series transcriptome analysis of the older adult mouse cochlea after noise exposure
After euthanasia, all cochleas were dissected using cold instruments under a binocular microscope. Next, they were rapidly put in RNAlater and stored at -80 C.  The noise signal was generated using RPvdsEX software (Tucker-Davis Technologies, Alachua, FL, USA). The signal was routed through an attenuator and power amplifier (XPS-1200, Gemsound, New York, NY, USA) to a high-frequency tweeter speaker (HTH 8.7, Visaton, Haan, Germany). The noise exposure was performed in a ventilated soundproof box while the animals were awake and continuously being monitored via a webcam. Before the mice were exposed to noise, the sound levels were calibrated at several locations in the soundproof box to ensure uniformity of the stimulus. Across these measured sites within the chamber, the noise intensity varied by less or equal to 2dB. During the noise exposure, the animals were placed in a cage in the box at the locations of calibration so that the stimulus intensity was known for each animal individually. Afterwards, total RNA was isolated using the Rneasy mini kit® (Qiagen, Benelux, Belgium), according to the manufacturer's protocol. Briefly, the cochlea was disrupted using a tissue homogenizer (Multipro, Dremel, Belgium) and lysate was homogenized with lysis buffer containing beta216 mercaptoethanol, followed by RNA extraction and purification using RNeasy mini spin columns. The concentration of the RNA samples was evaluated on the Qubit fluorometer 4.0, using the Qubit high sense RNA assay (Thermo Scientific, Wilmington, USA). The integrity and purity of the RNA samples was evaluated by the Agilent 4150 TapeStation System using the recommended RNA ScreenTape assay (Agilent Technologies, Inc., Waldbronn, Germany). For all samples, the RNA integrity number (RIN) values were above 7, indicating the superior quality of the isolated RNA. The RNA library preparation was performed using the QuantSeq 3' mRNA-Seq V2 Library Prep Kit FWD with UDI 12 nt Set B1, combined with the QuantSeq-Flex First Strand Synthesis Module V2 to create longer fragments (Lexogen, Vienna, Austria). Before the library amplification PCR, the optimal PCR cycles were evaluated with the PCR Add-on and Reamplification Kit V2 (Lexogen, Vienna, Austria). The finalized cDNA libraries concentrations were measured using the Qubit high sensitivity dsDNA kit (Qubit 4.0, Thermo Scientific, Wilmington, USA). The fragment lengths, distribution and the quality of the libraries were evaluated using the D1000 ScreenTape assay (TapeStation, Agilent Technologies, Inc., Waldbronn, Germany). All libraries were then equimolar pooled.  