*2.5. Transcriptomic Response Analysis*

An expected median concentration of 2.4 mg/L for pollutant mixtures was selected for transcriptomic analyses according to the EC50-48 and EC50-72 h values. The *P. tricornutum* cells were harvested at 48 and 72 h for subsequent transcriptome analysis. Specifically, 200 mL of *P. tricornutum* cultures were harvested, centrifuged, and stored at −80 ◦C until subsequent RNA extraction. Total RNA was isolated using the TRIzol reagent (Invitrogen, Carlsbad, CA, USA) according to the manufacturer's instructions, followed by an evaluation of RNA integrity and quality with the RNA Nano 6000 Assay Kit for the Bioanalyzer 2100 system (Agilent Technologies, Santa Clara, CA, USA). mRNAs were purified to construct cDNA libraries for sequencing conducted at Novogene (Beijing, China) on the Illumina NovaSeq platform (Illumina Inc., San Diego, CA, USA). According to the manufacturer's instructions, cDNA libraries were constructed with the NEBNext Ultra RNA Library Prep Kit for Illumina (New England BioLabs, USA).

Raw sequence reads were processed to obtain clean reads by removing adapters, N bases, and low-quality reads. Clean data were mapped to the *P. tricornutum* CCAP 1055/1 reference genome retrieved from the NCBI database. The reference genome index was constructed using Hisat2 v2.0.5, which was also used to align the paired-end clean reads to the reference genome. Genes were assigned to the Kyoto Encyclopedia of Genes and Genomes (KEGG) and the Gene Ontology (GO) databases for pathway and functional analyses. Raw reads were submitted to the Sequence Read Archive database under accession number PRJNA744053.

Differential expression analysis was performed using the DESeq2 R package (1.20.0). Thresholds including |fold changes| >2 and *p* < 0.05 were used to identify significantly differentially expressed genes (DEGs). DEG heatmaps were plotted using an online tool (https://www.omicshare.com/tools/, accessed on 29 September 2021).

#### **3. Results**
