Purified total RNA was first evaluated for its quantity and quality using an Agilent Bioanalyzer 2100. All RNA samples had a RIN (RNA integrity number) of 8 or higher. Ten nanograms of total RNA per sample was used for library preparation. Complementary DNA (cDNA) was first synthesized through RT with poly-dT priming, using the SMART-Seq v4 Ultra Low Input RNA Kit for Sequencing (Takara Clontech Laboratories Inc.), followed by cDNA shearing with Covaris AFA sonicator (Covaris) and size selection with AMPure beads (Beckman Coulter). Barcoded cDNA library was then prepared using the Ion Plus Fragment Library Kit (Thermo Fisher Scientific). Each library was quantified and its quality was assessed using Agilent Bioanalyzer, and multiple libraries were pooled in equal molarity. Average size of library insert was about 150 bp. Eight microliters of 100 pM pooled libraries was applied to ion sphere particle (ISP) template preparation and amplification using Ion OneTouch 2, followed by ISP loading onto a PI Chip and sequencing on an Ion Proton semiconductor (Thermo Fisher Scientific). Each PI Chip allows loading of about 140 million ISP templates, generating about 100 million reads up to 10 to 15 Gbp. A Phred quality score (Q score) was used to measure the quality of sequencing. More than 90% of the sequencing reads reached Q30 (99.9% base call accuracy).

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