RNA extraction and RNA sequence
tumor
TUMOR
Total RNA was extracted from snap-frozen tumor specimens. RNA purity was checked using the NanoPhotometer ® spectrophotometer (IMPLEN, CA, USA). RNA integrity was assessed using the RNA Nano 6000 Assay Kit of the Bioanalyzer 2100 system (Agilent Technologies, CA, USA). 1 µg RNA per sample was used as input material for the RNA sample preparations. Sequencing libraries were generated using NEBNext® UltraTM RNA Library Prep Kit for Illumina® (NEB, USA) following manufacturer’s recommendations and index codes were added to attribute sequences to each sample. Purified mRNA was randomly fragmented using divalent cations under elevated temperature in NEBNext First Strand Synthesis Reaction Buffer(5X). Using mRNA as template, double-stranded cDNA was synthesized by reverse transcription. cDNA fragments of around 200 bp were selected (AMPure XP beads) and purified (Agilent Bioanalyzer 2100 system) to ensure the quality of the library. The clustering of the index-coded samples was performed on a cBot Cluster Generation System using TruSeq PE Cluster Kit v3-cBot-HS (Illumia) according to the manufacturer’s instructions. After cluster generation, the library preparations were sequenced on an Illumina Novaseq platform and 150 bp paired-end reads were generated.