PAIRED ADJACENT NORMAL OF TY2T
Whole transcriptome libraries were prepared using Ion Total RNAseq v2 kit (Life Technologies, USA) according to manufacturers protocol. Two microgram of total RNA were used for ribosomal removal using RiboMinus Eukaryote kit (Life Technologies, USA). ERCC RNA Spike-In Control Mixes (Thermo Scientific, USA) were added into rRNA depleted-RNA before RNA fragmentation using RNase III. rRNA-depleted and fragmented RNA were quantified using Qubit fluorometer assay and Agilent RNA 6000 Nano Kit. Subsequently, the fragmented RNA was hybridized and ligated to adaptors, reverse-transcribed, purified, size-selected and amplified. The final libraries were quantified using Agilent High Sensitivity DNA chip (Agilent Technologies, USA) and were normalized to 100  130 pM for the next step. As for RNA sequencing, each library were subjected to clonal amplification on Ion Chef System using Ion PI IC 200 kit followed by sequencing using PI BC v2 chip on Ion Proton system (all from Life Technologies, USA). Two to three RNA libraries were pooled into a single chip.