Samples for deep sequencing analysis were prepared from 10 µg of each sample following Illumina's Small RNA sample preparation protocol (v1.5). During this process, samples were ligated with 3′ and 5′ adaptors, reverse transcribed and then amplified using PCR. Illumina Genome Analyzer IIx instrument (USA) was used for sequencing.
The data sequences were screened for the sequence of the small-RNA adaptor, and the adaptor sequences were trimmed using standard settings in Illumina's GAPipeline1.0. Processed Illumina data was managed by RandA software (Isakov et al., 2012 (link)). The reads were aligned to the human subset miRNAs in the miRbase database using BWA-aligner software. The number of reads was standardized by mapping each transcript according to its length and the initial total number of mapped reads in the sample based on the ‘reads per kilo-base per million’ (RPKM) method (Mortazavi et al., 2008 (link)). Only perfect matches were counted in the main analysis. Next, results were ranked in terms of differentially expressed miRNAs between the two samples. Statistical analysis was performed using a chi-square distribution.
The data sequences were screened for the sequence of the small-RNA adaptor, and the adaptor sequences were trimmed using standard settings in Illumina's GAPipeline1.0. Processed Illumina data was managed by RandA software (Isakov et al., 2012 (link)). The reads were aligned to the human subset miRNAs in the miRbase database using BWA-aligner software. The number of reads was standardized by mapping each transcript according to its length and the initial total number of mapped reads in the sample based on the ‘reads per kilo-base per million’ (RPKM) method (Mortazavi et al., 2008 (link)). Only perfect matches were counted in the main analysis. Next, results were ranked in terms of differentially expressed miRNAs between the two samples. Statistical analysis was performed using a chi-square distribution.
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