Name: W4RR-3C_s
Instrument: Illumina HiSeq 4000
Strategy: RNA-Seq
Source: TRANSCRIPTOMIC
Selection: PolyA
Layout: SINGLE
Construction protocol: The animals were anesthetized by intraperitoneal injection of 0.3% pentobarbital sodium and the body temperature was maintained by a heating pad. Five minutes before laser photocoagulation, compound tropicamide eye drops were used to disperse the pupil of both eyes. Eyes were anesthetized with two drops of 4 mg/mL oxybuprocaine hydrochloride eye drops (Santen Pharmaceutical Co., Ltd., Suzhou, China) and carbomer eye drops (Dr. Gerhard Mann, Chem.-Pharm. Fabrik GmbH, Germany)was used to prevent corneal dryness. After the animals were fixed, laser photocoagulation was performed at 1 PD around the optic disc, 8 spots in total. The laser wavelength was 647.1 nm, diameter of the spots was 50 μm, and the exposure time was 0.01–0.05. Energy of the laser was adjusted according to the retinal reaction. The effective spots were marked by the formation of bubbles without bleeding after photocoagulation. Eyes with ruptured capillaries small blood vessels were not used for subsequent experiments. Retinas and choroids were collected from the tree shrews after 7, 21, and 30 days of laser photocoagulation. Total RNA was extracted from the collected samples using TRIzol according to the standard protocol (Invitrogen, Carlsbad, CA, USA). Purity of the extracted RNA was spectrophotometrically quantified with NanoPhotometer (Implen, Westlake Village, CA, USA). RNA integrity was determined using Bioanalyzer 2100 system (Agilent Technologies, Santa Clara, CA, USA). In case the RNA content was insufficient(RNA < 2ug), the sample was mixed with other samples of the same group. First-strand DNA was synthesized using random primers and M-MuLV reverse transcriptase (RNase H-). Second-strand DNA was synthesized by DNA polymerase I and RNase H, wherein dTTPs were replaced by dUTPs. Further, the dU-containing second-strand cDNA was degraded by the USER enzyme, and the cDNA was PCR-amplified to obtain a library. The cDNA was quantified using Qubit 2.0, In addition, insert size of the library was determined by diluting the library to 1 ng/μL and subjecting to Agilent 2100 system. Effective concentration of the cDNA library was quantified accurately by qPCR (effective library concentration > 2 nM) to ensure quality of the cDNA library.