miRNA Sequencing and Expression Analysis

For miRNA sequencing, 5 µg of total RNA form each sample was ligated with both a 5′ adapter and 3′ adapter for reverse transcription using Superscript II at 42°C for 1 h and 70°C for 15 min. Subsequently, the reverse transcribed products were amplified using the following PCR program: a 15-cycle reaction at 98°C for 30 sec, followed by 15 cycles of 98°C for 10 sec, 72°C for 15 sec, and then 72°C for 10 min. After obtaining a ∼92-bp DNA band on 6% PAGE gels, the PCR products were ethanol precipitated and purified using Spin-X filter columns. Finally, miRNA libraries were sequenced on the Illumina Cluster Station and Genome Analyzer II following the manufacturer's protocol.
Low quality reads were trimmed and adapter sequences were accurately clipped with the aid of a dynamic programming algorithm before subsequent statistical analysis. After elimination of the duplicate reads, the remaining reads of at least 18 nt were mapped to a human reference genome (hg19) using SOAP V2.0. To remove tags originating from protein-coding genes, repeat sequences, rRNA, tRNA, snRNA, and snoRNA, we also mapped the short read tags to UCSC RefGene, RepeatMasker and NCBI Refseq, as well as our in-house ncRNA annotation datasets compiled from the NCBI GenBank database (http://www.ncbi.nih.gov). The same pipeline used for DGE mRNA differential expression analysis was also used for miRNA expression analysis.