Instrument: Illumina HiSeq 2000
Strategy: ncRNA-Seq
Source: TRANSCRIPTOMIC
Selection: size fractionation
Layout: SINGLE
Construction protocol: Total RNA was extracted from ISE6 cells using Trizol-LS (Invitrogen) according to the manufacturer's instructions.For small RNA libraries of ISE6 cells depleted of small RNA factors and their control samples, 1ug total RNA was used for library construction. For 5'-tri-P libraries, the small RNA fraction (~15-35nt) was isolated by gel extraction and RNA species bearing 5'-OH were monophosphorylated by T4 polynucletide kinase (NEB). This was followed by treatment by a terminator exonuclease (Epicentre), dephosphorylation by Calf Intestine Phosphatase (NEB) and re-phosphorylation by T4 polynucletide kinase (NEB). For the oxidized small RNA library, small RNAs (~15-35nt) from 50ug total RNA was isolated by gel extraction and treated with 25mM NaIO4 dissolved in 60mM Borax buffer and incubated for 30 minutes at room temperature in the dark. For total RNAseq analysis, three sets of knockdown experiments were performed independently, and total RNA samples extracted by Trizol-LS (Invitrogen) were sent to BGI (Hongkong) for ribosomal RNA depletion using Ribo-Zero Gold rRNA Removal Kit (Human/Mouse/Rat) (Epicentre). For small RNA libraries of ISE6 cells depleted of small RNA factors and their control samples, 1ug total RNA was used for library construction using the TruSeq Small RNA Library Preparation kit (Illumina). Both 5'-tir-P enriched and oxidized samples were subjected to a small RNA library construction by the TruSeq Small RNA Library Preparation kit (Illumina). For total RNAseq analysis, library construction used non-stranded (Replicate 1) or stranded (Replicates 2 and 3) TruSeq mRNA Library Prep Kit (Illumina).