Transcriptome Sequencing and Differential Expression Analysis

Transcriptome sequencing was performed using the high-throughput sequencing platform of Illumina HiSeq 4000 (Illumina, San Diego, CA, USA). 5 µg of total RNA was used for RNA-seq analysis. Base calling was adopted to convert original sequencing images to sequential data. The raw reads were subjected to adapter trimming and low quality filtering using Trimmomatic program [47 (link)]. The high quality clean reads were aligned to the human genome using TopHat [48 (link)]. Human genome sequence and gene annotation were obtained from the UCSC Genome Website. Cuffdiff was used to profile differentially expressed genes with default parameters [49 (link)]. All unigenes were queried against commonly used databases using BLASTx search to identify homologs (E-value < 10⁻¹⁰). The databases used were Swiss-prot [50 (link)], KEGG [51 (link)], Nr [52 (link)], KOG [53 (link)], and GO [54 (link)]. The differentially expressed genes (DEGs) between control and phycocyanin-treated A549 cell line were identified based on fragments per kilobases per millionreads (FPKM) using RSEM 1.2.31 [55 (link)]. DESeq was used to determine the FDR (false discovery rate) threshold (adjust p value). If the FDR was less than 0.05 in the multi-group comparison, it was considered to be a significantly different expression level.