Sequencing control studio software

The serum samples of 3 AIED-1 and 3 control mice were obtained and subjected to miRNA deep sequencing analysis (LC Sciences). Briefly, total RNA was extracted from serum using TRIzol (Thermo Fisher Scientific, Inc.) and ligated to 3′ and RNA 5′ adapters of RNA at 70°C for 2 min each. The total RNA was reverse transcribed into cDNA using the SuperrScript II Reverse Transcriptase (Invitrogen; Thermo Fisher Scientific, Inc.) at 50°C for 1 h. PCR amplification was run with 25 µl reaction mixture, including 11.5 µl cDNA, 12.5 µl Phusion^® Hot Start Flex 2X Master Mix (New England Biolabs, Inc.) and 0.5 µl primers (each, 5′-GATCGGAAGAGCACACGTCTGAACTCCAGTCACAAGACGGAATCTCGTATGCCGTCTTCTGCTTG-3′; 5′-AGATCGGAAGAGCGTCGTGTAGGGAAAGA-3′). PCR amplification was carried out under conditions of 98°C for 3 sec followed by 12 cycles of 98°C for 10 sec, 60°C for 30 sec, and 72°C for 15 sec, and 72°C for 10 min. Small cDNA fractions were isolated by using 6% Tris-borate-EDTA/polyacrylamide gel electrophoresis (TBE-PAGE). Subsequently, the cDNA constructs were purified and the library was validated. Finally, the purified PCR products were used for sequencing analysis via the Illumina Solexa Sequencer (Illumina, Inc.). Raw sequencing reads were obtained using Illumina's Sequencing Control Studio software (v2.8; Illumina, Inc.).