RNA-seq of mus musculus: 10 weeks male retina replicate 3
Total RNA was extracted from retina samples by using TRIzol reagent according to the manufacturer's instructions, including treatment with DNase. RNA purity was checked using the NanoPhotometer spectrophotometer, and samples with an optical density 260/280 and 260/230 ratio between 1.8 and 2.1 were used. RNA integrity was assessed using the RNA Nano 6000 Assay Kit of the Bioanalyzer 2100 system to calculate an RIN. Only RNA samples with a RIN >8 were used for sequencing analysis.  After removing rRNA, total RNA was break into short fragments of 250-300 bp. The fragmented RNA was used as a template and random oligonucleotides were used as primers to synthesize the first strand of cDNA, then RNA templates were degraded with RNase H. The second strand of cDNA was synthesized in DNA polymerase I system with the first strand of cDNA as templates and dNTPs as raw materials. The double-stranded cDNA got through end repair, adding A-tail and adaptors, and the cDNAs of about 200 bp were selected using AMPure XP beads. The second strand of the cDNAs containing dUTPs were digested using 3 l USER enzyme at 37C for 15 min followed by 5 min at 95 C, and finally PCR was performed to amplify and construct the library. After library construction, initial quantification was performed using Qubit 2.0 and dilute the library to 1.5 ng/ul, followed by detection of the insert size of the library using Agilent 2100. Then the effective concentration of the library (> 3 nM) was accurately quantified using qRT-PCR. After passing the library qualification, the library was pooled and sequenced by Illumina PE150, and the insert cDNA was the unit directly sequenced in the constructed library. The raw image files are converted into sequenced reads by CASAVA (v1.8) base identification and stored in FASTQ format. Raw data (raw reads) of FASTQ format were firstly processed through in-house perl scripts. During this process, clean data  were obtained after removing low-quality reads (> 50% of bases with sQ <5) and reads containing adapter or ploy-N (> 21% of uncertain base) from raw data.