RNA-seq of Immortalized RAFLS treated with TNFα for 24h and DMSO or celastrol derivatives for 4h in human

This experiment is to explore the mechanism of anti-inflammatory activity of celastrol derivatives on Rheumatoid arthritis fibroblast-like synoviocytes (RAFLS), compared with celastrol. Immortalized RAFLS cells were simulated with 1 ng/mL TNFα for 24h, and exposed to different treatments during the last 4 hours. Total RNA was extracted with Trizol. Transcriptomic profiling was performed with an in-house, proprietary method that captures the 3’ primed end of mRNA molecules for sequencing. The data was processed with a custom script. Briefly, sequencing reads were demultiplexed based on sample barcode with idemux, trimmed with cutadapt to remove polyA, adaptor and low-quality sequences, mapped to human GRCh38 reference assembly with STAR aligner. PCR duplicates were removed based on UMI with UMI-tools. Htseq-count was used to generate read counts. The count matrix was analyzed with DESeq2 package to determine differential genes. Enrichment and GSEA was performed with clusterprofiler. DMSO: negative control, 3 samples. Cel: celastrol, 3 samples. Cel5, celastrol derivative 5, 3 samples. Cel6: celastrol derivative 6, 3 samples. All cells are simulated with 1 ng/mL TNFα for 24h, and exposed to different treatments during the last 4 hours.