Effect of Aeroplysinin-1 on gene expression in Murine RAW264.7 macrophages in basal and LPS stimulated conditions (2hours)

To determine the effect of Aeroplysinin-1 (Ap) on gene expression, we treated murine RAW264.7 macrophages (ATCC TIB-71) with DMSO control or Aeroplsinin-1 (1uM) for 2 hours. Unstimulated and LPS stimulated (0.1ug/mL) conditions were investigated. Three biological replicates were used per group for RNASeq analysis. Derivatives of Aeroplysinin-1 were also tested (Ap7, Ap-9) at the same concentration and time points. FPKM as well as DGE files are provided. The title of each DGE file explains which groups are being compared. Word document (RNASeq analysis documentation) describes methods of processing reads.Lysates were prepared by washing cells twice with PBS+, lysed in recommended volume of RLT Lysis buffer from the Qiagen RNeasy RNA Isolation Kit (Cat. No. / ID: 74104) and collected by scraping with a sterile cell scraper. Lysates were vortexed for 20 seconds, centrifuged at max speed for 3 minutes at room temperature at stored at -80C for up to two weeks prior to extraction according to manufacturer’s instructions. RNA samples were submitted to the Center for Applied Genomics Next Generation Sequencing Facility at SickKids for quality control on the bioanalyzer, library prep and sequencing. Samples had an Agilent Bioanalyzer RNA integrity number above 7 and an OD260/230 ratio of 2.0-2.2. Library prep was performed using the NEB Ultra II Directional mRNA kit. Sequencing was performed with the Illumina Hiseq2500 platform with high throughput flowcell PE2x125bp (paired end 2x125 base pair configuration), with 25-30 million reads per sample.For RAW264.7 cells, reference-based count-based differential gene expression with STAR+Htseq+DESeq2, comparing the following groups; (1) DMSO no LPS vs Ap no LPS, (2) DMSO no LPS vs Ap7 no LPS, (3) Ap7 no LPS vs Ap no LPS, (4) Ap no LPS vs Ap9 no LPS, (5) DMSO LPS vs Ap LPS, (6) DMSO LPS vs Ap7 LPS, (7) Ap7 LPS vs Ap LPS and Ap LPS vs Ap9 LPS. The library was a standard paired end library, the genome reference sequence was GRCh38, Gencode annotations, Release 36 for RAW264.7 cells.