GSM8069432: Mock-Infection-1; Homo sapiens; RNA-Seq
The samples were extracted by a modified Folch extraction procedure. Specifically, 4.9 ml of a Chloroform/Methanol solution (2:1, v/v) was added to the cells. The mixture was subjected to two cycles of sonication at 10 microns for 20 seconds on ice, followed by a 10 second pause. Subsequently, the mixture was centrifuged at 4000 rpm for 5 minutes, resulting in the formation of a clear supernatant. To this, 2.5 ml of the supernatant was combined with 0.5 ml of NaCl/Water (0.73%, w/v) and vortexed for 30 seconds. The resulting aqueous layer was discarded, and the organic layer was washed twice with 0.5 ml of Methanol/Water (1:1, v/v) without mixing. The aqueous layer was again discarded, and the organic layer was dried under a gentle stream of nitrogen at 45°C. For esterification, the dried sample was dissolved in a mixture of 0.1 ml of chloroform, 1 ml of methanol, and 50 µl of concentrated hydrochloric acid (35%, w/w). The solution was then covered with nitrogen and the tube was tightly closed. After vortexing, the tube was heated at 100°C for 1 hour. Once the solution cooled to room temperature, 1 ml of hexane and 1 ml of water were added for extraction of fatty acid methyl esters (FAMEs). The tube was vortexed, and after phase separation, up to 1 µl of the hexane phase was injected for GC-MS analysis. Sequencing libraries were constructed and sequenced in the Proteomics and Metabolomics Core (LKS Faculty of Medicine, The University of Hong Kong). After filtering the low-quality reads, clean reads were mapped to reference sequences using HISAT/Bowtie2. The DEGs in treated samples were submitted to DAVID server for pathway enrichment and cluster analysis. Gene Ontology (GO) enrichment analysis of DEGs was implemented using the GOseq R package. KOBAS software was used to examine the statistical enrichment of DEGS in The Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways.