Identification and characterization of known and novel microRNAs in spleen tissues of Chinese giant salamander base on deep sequencing approach
Small RNA library construction was conducted by using Illumina TruSeq Small RNA Preparation Kit following the manufacturer′s instructions (LC Sciences, Hangzhou, China). Briefly, the total RNA was ligated to RNA 3′ and RNA 5′ adapters. And then, reverse transcription followed by PCR was performed to create cDNA constructs based on the small RNAs ligated with 3′ and 5′ adapters. Small cDNA fractions that range from 18 nt to 30 nt in length were isolated by using 6% denaturing polyacrylamide gelelectrophoresis. Subsequently, cDNA construct was purified and the library was validated. Finally, the purified PCR products were used directly for cluster generation and sequencing analysis using the Illuminas Solexa Sequencer according to the manufacturer′s instructions. Raw sequencing reads were obtained by using related Illumina′s analysis software. The ACGT101-miR program (version 4.2; LC Sciences) was used for standard sequencing data analysis.