NextSeq 500 sequencing; Dysfunction of retinal dopaminergic system exacerbates experimental autoimmune uveoretinitis in Gnat1-deficient mice
The collection of neuroretina samples was carried out in a dark room with dim red light. Neuroretinas were excised from enucleated eyes using a binocular stereomicroscope with a far-red light source. The eyeball was punctured with a 30 G needle in the anterior part of the pars planar, a small microdissecting scissors were then inserted into this puncture, and the entire anterior portion of the eye was cut off and the lens was pushed out. The neuroretina was lifted from the optic nerve head, pinched off at the point where it connects to the optic nerve, and gently pulled away from the eye cup with forceps. The vitreous humor and remnants of the retinal pigment epithelium were separated from the neuroretina and discarded. Neuroretinas were snapped frozen on dry ice and stored at -80°C for RNA extraction. Mice were first dark adapted overnight and then killed by an overdose of ketamine and xylazine (4 mg and 0.8 mg) administered intraperitoneally, followed by cardiac perfusion with 1x PBS in a dark room with dim red light. The eyes of the sacrificed mice were carefully removed and immersed in saline (0.9% sodium chloride) in a Petri dish placed on ice for neuroretina collection. Total RNA was isolated from neuroretinas using RNeasy Mini Kit (Qiagen, Hilden, Germany) according to the manufacture's recommendation with on-column DNase digestion step using RNase-Free DNase Set (Qiagen) to eliminate genomic DNA. The sequencing libraries were prepared using the SMARTer Stranded Total RNA-Seq Kit v2 (Takara, Shiga, Japan) following the manufacturer's instructions.