GSM9068777: RNA-seq sequencing data 2 of mice liver tissues with DEHP exposure; Mus musculus; RNA-Seq
Total RNA was isolated from mouse liver tissues using TRIzol (Beyotime); mRNA was purified from total RNA using poly-T oligo-attached magnetic beads. Briefly, total RNA was isolated from mouse liver tissues using TRIzol (Beyotime). RNA concentration and integrity were assessed using a Nanodrop photometer and an Agilent 5400 bioanalyzer. Sequencing libraries were prepared using the NEBNext Ultra Directional RNA Library Prep Kit for Illumina in accordance with the manufacturer's recommendations. First, mRNA was purified from total RNA using poly-T oligo-attached magnetic beads and subsequently fragmented in NEBNext First Strand Synthesis Reaction Buffer with divalent cations. The first cDNA strand was synthesized using random hexamer primers and M-MuLV Reverse Transcriptase (RNase H-). The synthesis of the second cDNA strand was performed with DNA polymerase I, where all dTTPs in the reaction buffer were replaced by dUTPs. The remaining overhangs were converted into blunt ends through exonuclease and polymerase activities. Following the adenylation of the 3' end of the DNA fragments, NEBNext Adaptors with a hairpin loop structure were ligated. Next, 3 μl of USER Enzyme (NEB, USA) was incubated with the size-selected, adaptor-ligated cDNA at 37 °C for 15 min, followed by a 5-min incubation at 95 °C. PCR amplification was then performed using Phusion High-Fidelity DNA polymerase, universal PCR primers, and index primers. The PCR products were purified using KAPA Pure Beads. Library quality was assessed with the Agilent 5400 system (Agilent, USA). Paired-end sequencing of the samples was performed on the Illumina platform (Illumina, USA)