Effect of C2S in RAW264.7 murine macrophages

This study aimed to investigate the mechanism of C2S-induced macrophagic inflammation and its’ effect on osteogenic differentiation of precursor cells. This study used the C2S (75-150 μg/ml) extract to induce macrophagic inflammation in RAW264.7 murine macrophages. RNA sequencing was preformed for analyzing mechanism of mitochondrial function and autophagy. The macrophages were treated with and without C2S (150 µg/ml) for 24h were used for RNA sequencing. Total RNA was extracted by using the Trizol kit (Invitrogen, Carlsbad, CA, United States), according to the manufacturer’s protocol. The RNA concentration was determined using Qubit, and the RNA amount and purity of each sample was assessed with a NanoDrop spectrophotometer. RNA was isolated in 40 µl of DEPC water and stored at -80. RNA-seq libraries were prepared by using the Illumina TruseqTM RNA sample prep Kit and were sequenced using an Illumina HiSeq.Low-quality base and adapters were removed by using “fastp”. Clean reads were aligned to the mouse genome (mm10) reference genome using STAR. Count the number of reads mapped to each gene by using RSEM. And the "edgeR" R software package was used to calculate the differential expression analysis between the two groups. KEGG analysis was performed using the “clusterProfiler” R package. KEGG terms with corrected p < 0.05 were considered significantly enriched. contributor: National Collection of Authenticated Cell Cultures, TCM13, Shanghai, China