Transcriptional profiling of visual stimulation-activated mouse retinal cells using RNA-Seq
Transcriptional profiling of visual stimulation-activated mouse retinal cells using RNA-Seq
After 8 hours of visual stimulation on the last day of the experiment, all mice were sacrificed by rapid cervical dislocation. The eyes were dissected and immediately processed for the collection of neural retina samples. The eye was punctured with a 30 G needle in the pars plana; a small microdissecting scissors were inserted into this puncture; and the entire anterior segment of the eye was cut off and the lens was pushed out. The neural retina was gently pulled away from the posterior eye cup (containing retinal pigment epithelium, choroid and sclera) and placed into ice-cold homogenization buffer. Ten neural retinas from five mice were pooled as one sample in both stimulated and control groups and homogenized with a Teflon-glass tissue grinder. The homogenates were then immunoprecipitated with an antibody to pS6 Ser244/247 (#2215, Cell Signaling Technology, Danvers, MA, USA). Mice were fed ad libitum and kept on a 12-hour light/dark cycle. Visual stimulation was performed in optomotor drums for 14 days during the whole light phase. In the optomotor drums, mice were exposed to vertical black and white stripes rotating with a spatial frequency of 0.2 cycles per degree and alternating clockwise and counterclockwise every 20 s. RNA was extracted from pS6 immunoprecipitates using the RNeasy Micro Kit (Qiagen, Hilden, Germany) according to manufacturer's instructions. The sequencing libraries were prepared using the SMARTer Stranded Total RNA-Seq Kit v2 (Takara, Shiga, Japan) following the manufacturerâ€™s instructions.