Illumina HiSeq 4000 sequencing; Redox activation of excitatory pathways in auditory neurons as mechanism of age-related hearing loss
Redox activation of excitatory pathways in auditory neurons as mechanism of age-related hearing loss
Cochlea were quickly dissected in cold PBS, carefully removing remaining blood and surrounding tissues, immediately frozen in liquid nitrogen and kept at -80Â°C for further RNA extraction. Cochlea were physically homogenized with clean steel balls and tissueLyser (Qiagen) for 30 seconds at 30rpm, and the DNA and RNA precipitated with 750ul Trizol. After adding 150ul chloroform, (shaking, resting and centrifuging step) we collected the aqueous phase and performed column purification with Qiagen RNeasy Micro kit, ignoring the lysis and homogenization steps described in the kit protocol (adding directly 70% ethanol). TruSeq ribodepleted stranded mRNA from 6 weeks old A/J mice (3 WT vs. 3 p22phox deficient) was applied to eliminate ribosomic RNA and sequenced using Illumina TruSeq protocol.