The RNA extraction protocol, Qiagen MiRNeasy Mini (cat no. 217004) protocol, has been validated to be effective to purify both total RNA and miRNA37–42. For each sample, the RIN score was measured using the Agilent Bioanalyzer Eukaryotic Total RNA Nano chip. Those RNA samples with RIN score of 5 or better were submitted for cDNA library construction. RIN score affects the fragment lengths of RNA inserts for library construction, and therefore we batched samples according to RIN scores so that library pools would have uniform insert sizes. In order to get correct alignment, we trimmed the reads if they have low quality bases (Q10) from beginning and end or those reads derived from adapters or rRNA sequences. Experimental replicates and controls were designed to calibrate data.
