title: T15-10_2
design: Total RNA was extracted with Trizol (Invitrogen) and assessed with Agilent 2100 BioAnalyzer and Qubit Fluorometer (Invitrogen). Total RNA samples that meet the following requirements were used in subsequent experiments. The triplicate samples of all assays were constructed an independent library, and do the following sequencing and analysis. The NEB Next Ultra RNA Library Prep Kit for Illumina (NEB) was used to construct the libraries for sequencing. NEB Next Poly(A) mRNA Magnetic Isolation Module (NEB) kit was used to enrich the poly(A) tailed mRNA molecules from 1 g total RNA. The mRNA was fragmented into ~200 base pair pieces. The first-strand cDNA was synthesized from the mRNA fragments reverse transcriptase and random hexamer primers, and then the second-strand cDNA was synthesized using DNA polymerase I and RNaseH. The end of the cDNA fragment was subjected to an end repair process that included the addition of a single A base, followed by ligation of the adapters. Products were purified and enriched by polymerase chain reaction (PCR) to amplify the library DNA. The final libraries were quantified using KAPA Library Quantification kit (KAPA Biosystems, South Africa) and an Agilent 2100 Bioanalyzer. After quantitative reverse transcription-polymerase chain reaction (RT-qPCR) validation, libraries were subjected to paired-end sequencing with pair end 150-base pair reading length on an Illumina NovaSeq sequencer (Illumina).