Casava pipeline v2

Seventeen indexed miRNA libraries were prepared using the Illumina Truseq Small RNA Library Preparation Kit (Illumina Inc., San Diego, CA, USA). Procedure for the preparation of the sequencing libraries were performed according to the manufacturer's protocol. Briefly, a 3′-adaptor sequence was first added to the 3′-end of the small RNA molecule then a 5′ adaptor sequence was added to the 5′-end of the small RNA. The adaptor ligated RNA was then reverse transcribed into cDNA, which was eliminated by adding RNase. The libraries were separated from adapter dimers by size fractionation in 8% TBE polyacrylamide gel (Life Technologies, Carlsbad, CA, USA). The 150 bp small RNA libraries were excised and purified from the gel. We then used the Agilent High Sensitivity DNA Kit (Agilent Technologies, Colorado Springs, CO, USA) to quantify the molarity and confirm the size distribution. The 17 Indexed miRNA libraries were pooled in equimolar concentrations and sequenced on one lane of an Illumina HiSeq 2000 platform (Illumina Inc., San Diego, CA, USA). The fastq files were produced using the CASAVA pipeline v2.0 (Illumina Inc., San Diego, CA, USA) and all generated sequence were deposited in NCBI dbGAP (accession number: phs001282.v2).