Endogenous reference gene selection and primer design

According to the published Chlorella sp.TLD 6B transcriptome sequencing data (NCBI GEO: {"type":"entrez-geo","attrs":{"text":"GSE162916","term_id":"162916"}}GSE162916), 9 endogenous reference genes (based on their FPKM and fold change values) were selected²⁴. Chlorella sp. TLD 6B was evaluated by full-length transcriptome sequencing using PacBio instrument. Will get to the transcription of this sequence with TransDecoder after redundancy (https://github.com/TransDecoder/TransDecoder/Releases) to identify transcription candidate in this sequence coding region, Chlorella sp. TLD 6B CDS library is obtained. BLAST software (version 2.2.26) was used to compare the obtained non-redundant transcript sequences with NCBI non-redundant protein (NR), Swissprot (http://www.expasy.org/sprot/), GO (http://www.geneontology.org), COG(http://www.ncbi.nlm.nih.gov/COG/), Pfam (http://pfam.xfam.org/), KOG and KEGG databases (http://www.genome.jp/kegg) to obtain the annotation information of CDS database⁶⁴. Primers for a total of nine genes, 18S, CYP, EF-1α, GAPDH, GTP, IDH, UBC, α-TUB, and β-TUB were designed using Primer Premier 5(Premier, Canada) software according to Information about the CDS library and RT-qPCR primer design principles, and Sangon Biotech (Shanghai, China) synthesized the corresponding primers (Table ​(Table11).