RNA - Seq of C57BL mice: TCA - stimulated BMDM (M2 - type macrophages)
Healthy male C57BL/6 mice (6 - 8 weeks old, 20 - 25 g) were sacrificed by cervical dislocation, immersed in 75% ethanol for 5 min, then tibia and femur were obtained and placed in ice - cold PBS after related tissue removal. Bone marrow cells were flushed out with DMEM, filtered, and treated with red blood cell lysis buffer before being cultured in complete DMEM with GM - CSF. After M2 induction and TCA stimulation, cells were trypsinized, collected, and stored with Trizol.Total RNA was extracted using TRIzol, purified by centrifugation and isopropanol precipitation. RNA concentration and purity were measured by NanoDrop 2000. mRNA was enriched with Oligo(dT) - coated magnetic beads, fragmented, and then reverse - transcribed into cDNA. Adapter was ligated to the cDNA ends, followed by PCR amplification and purification to obtain the final sequencing library. High - throughput sequencing was performed on NovaSeq X Plus or DNBSEQ - T7 platform. Raw data were quality - assessed, low - quality reads were removed, and clean reads were aligned to the reference genome for expression and functional analysis.