mVenus overexpressing line4
Frozen pellets were lysed by pipetting into 1.5 mL of PureZol reagent (Cat. #7326890, Bio-Rad, United States) at room temperature, incubated for five minutes, then mixed with 300 L chloroform and kept for mixing on a rotor wheel for 15 minutes. The samples were centrifuged at 12,000  g for 15 minutes at 4 C, and 650 L of the upper aqueous phase was combined with an equal volume of 75% ethanol and loaded onto a Qiagen RNeasy Mini column (74,106, QIAGEN, Netherlands), where RNA was purified according to the manufacturers protocol. RNA concentration and purity were measured on a NanoPhotometer N50/N60 (Techno Scientific, United StatesUSA), and integrity was checked by electrophoresis on a 1% agarose gel. Sequencing libraries were prepared using the NEBNext Ultra II RNA Library Prep Kit (E7770L, NEB, United States), and their fragment size distributions were verified on a Fragment Analyzer before loading onto an Illumina NovaSeq 6000 for high-throughput paired-end sequencing. Raw sequencing reads underwent quality and adapter trimming with Trim Galore, and pre- and post-trim quality metrics were generated by FastQC using default parameters. Cleaned reads were aligned to the P. tricornutum reference genome ASM15095v2 (RefSeq GCF_000150955.2) using HISAT2, and resulting BAM files were imported into SeqMonk with default settings (retaining duplicates). Differential expression analysis was carried out in DESeq2 with BenjaminiHochberg adjustment; genes showing an adjusted p-value below 0.01 were defined as differentially regulated.