*2.4. RNA-Seq Analysis*

RNA sequencing (RNA-Seq) was conducted in collaboration with the Decode Genomics (Nanjing, China). Two biological replicate cell treatments were performed for each cell and nanoparticle. Totally, 12 RNA samples (two independent RNA samples of blank HL60 and KG1a, FeNP-treated HL60 and KG1a, and PBNP-treated HL60 and KG1a) were used to perform RNA-Seq analysis. The total RNA was isolated using the TRIzol® Reagent Kit (Thermo Fisher Scientific, Waltham, MA, USA) following the manufacturer's instructions. The degradation and contamination status of RNA were analyzed by 1% agarose gel electrophoresis, and the RNA purity was evaluated by Nanodrop 2000 according to the ratio of OD260nm/OD280nm (around 1.8–2.2). The RNA concentration was accurately quantified by Qubit (≥500 ng/μL), and the insert size of the library was assessed with Agilent 2100 to judge RNA integrity. After the library quality control was qualified, Illumina sequencing was performed using a paired end 150 bp (paired end, PE150) strategy. To ensure the quality of information analysis and clean reads, the raw reads obtained by sequencing were filtered to remove dirty reads that contained adapters, excessive N (≥10%, N: bases information cannot be determined), and a large number of low-quality bases. Subsequent analysis was based on clean reads. The HISAT2 software was used to align clean reads with the reference genome.
