Small rna v1.5 sample preparation guide

Total RNA was extracted from primary HACs and small RNAs enriched from 10 μg total RNA using the mirVana^™ miRNA Isolation Kit. The small RNA library was prepared using the Illumina Small RNA V1.5 Sample Preparation Guide, however sRNA adaptors were substituted with High Definition (HD) adaptors¹⁹ (link). Approximately 200 ng RNA enriched for small RNA was ligated to adenylated 3′ HD adaptor with truncated T4 RNA ligase 2 (New England Biolabs). The ligated fragment was then ligated to 5′ HD adaptor using T4 RNA ligase 1 (New England Biolabs). The ligated fragment was reverse transcribed followed by PCR amplification and size fractionated on an 8% (w/v) PAGE gel. A band corresponding to 145–150bp was gel purified and analysed on an Illumina Genome Analyzer IIX with 50 nt read length (Baseclear, Netherlands). Reads were trimmed for 4 nt barcodes on both ends and for Illumina adaptors on the 3′ end. Resulting reads longer than 16 nt were mapped to the human genome (version GRCh38) using Patman software²² (link), no mismatches were allowed. Reads mapping to more than 100 loci were discarded. The remaining reads were inputted to miRCat²³ (link) with default parameters. miRCat novel miRNA candidates were separated from known miRNAs using in-house scripts.