2.5.1. RNA Extraction and High-Throughput Sequencing

The Jurkat T cells were seeded in a 75 cm<sup>2</sup> cell culture bottle with 60 mL of medium at an initial concentration of 1.2 × <sup>10</sup><sup>7</sup> cells/bottle and treated for 36 h with DMM at final concentrations of 4 mg/L (EC10) and 21 mg/L (EC25). The cells were collected for transcriptome analysis. Total RNA was isolated using the TRIzol® reagent (Thermo Fisher, Waltham, MA, USA), according to the manufacturer's protocol, and genomic DNA was removed using DNase I (TaKaRa, Dalian, China). Then, RNA quality was determined by a 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA, USA) and quantified using an ND-2000 (NanoDrop, Wilmington, DE, USA). Only high-quality RNA samples (OD260/280 = 1.8~2.2, OD260/230 ≥ 2.0, RIN ≥ 6.5, 28S:18S ≥ 1.0, >1 μg) were used to construct a sequencing library.

The RNA transcriptome library was prepared following the TruSeqTM RNA sample preparation kit. Libraries were size selected for cDNA target fragments of 300 bp on 2% Low Range Ultra Agarose, followed by PCR amplification using Phusion DNA polymerase (NEB, Ipswich, MA, USA) for 15 PCR cycles. After quantification by TBS380, a paired-end RNA sequencing library was obtained by using a Nova Seq 6000 sequencer (2 × 150 bp read length).
