mRNA-Seq Library Preparation Protocol

For library construction, mRNA was purified from 3 μg of total RNA of each sample using poly-T oligo-attached magnetic beads (New England Biolabs) [27 (link)]. Transcriptome sequencing libraries were generated using an Illumina NEBNext® UltraTM RNA Library Prep Kit (New England Biolabs) according to the manufacturer’s instructions and index codes were added to attribute the sequences to the corresponding sample. Briefly, purified mRNA was cut into fragments and the cleaved mRNA fragments were reverse-transcribed into first-strand cDNA using random hexamers, followed by synthesis of double-strand cDNA. After blunting ends, the 250 to 300 bp fragments were purified using the AMPure XP system (Beckman Coulter, Brea, CA, USA). The purified cDNA fragments were then linked using an NEBNext Adaptor (New England Biolabs) with a hairpin loop structure and amplified by PCR. The AMPure XP system (Beckman Coulter) was used to purify the PCR products and the sample library quality was assessed using an Agilent Bioanalyzer 2100 system.