Total RNA was extracted using Trizol (Invitrogen, United States) from Chlorella cells (WT and LOG OX1, three biological replicates each) as per the manufacturer’s protocol. RNA-sequencing libraries were prepared with Illumina-compatible NEB Next® UltraTM II Directional RNA Library Prep Kit (New England Bio Labs, United States) at Genotypic Technology Pvt. Ltd., Bangalore, India, following manufacturer’s instructions. The Illumina-compatible sequencing library was quantified by Qubit fluorometer (ThermoFisher Scientific, United States), and its fragment size distribution was analyzed on Agilent 2200 tape station. The average fragment size across the libraries was observed to be 500 bp, with an average Qubit-based concentration of 11.6 ± 3 ng/μl. The samples were molar normalized and pooled for multiplexed paired-end sequencing on Illumina HiSeq X Ten sequencer. A total of 35.6 Gbp were generated across the six samples sequenced, with a mean quality score > 37, and >88% bases called with Phred score Q > 30. The raw data generated was checked for the quality using FastQC22. Reads were pre-processed to remove the adapter sequences and removal of the low-quality bases (<q30). Pre-processing of the data is done with Trimgalore3.3 HISAT-24, which is a splice aligner (Kim et al., 2015), was used to align the high-quality reads to the reference Chlorella variabilis NC64A genome4 downloaded from NCBI database with the default parameters. HTSeq was used to estimate and calculate gene abundance (Anders et al., 2015). Absolute counts for each gene were identified, which were used in differential expression calculations. DESeq was used to calculate the differentially expressed genes (DEGs) (Anders and Huber, 2010). Genes were categorized into up- and downregulated based on the fold change cut off of 1.5, adjusted p-value (q-values) ≤ 0.05. For each gene, gene ontology (GO) was assigned based on the homology search against algae reviewed protein sequences downloaded from the Uniprot database. Reference sequences were matched against Uniprot data using the Diamond BLAST program (Altschul et al., 1990). These GO terms were mapped to the differentially expressed (DE) genes. Pathways for each gene were obtained from the KEGG KAAS 5 server (Moriya et al., 2007). Compiled pathways per gene were mapped to the DEGs. The RNA-Seq, raw and processed files have been deposited to GEO database, NCBI with accession number GSE162985.

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