Figure 4
Methodology and validation of TRAP in MacTRAP mice. (A) Schematic illustrating the principle strategy and procedure of TRAP. MacTRAP kidneys are harvested, immediately homogenized and lysates subjected to immunoprecipitation (IP) with anti-eGFP-antibody-coated magnetic beads. Magnetic beads bind only to polysomes with the eGFP-tagged L10a protein (green). These bound polysomes represent the cell-specific (bound) fraction, which contains highly enriched RNA messages of renal macrophage origin. The unbound fraction represents RNA messages of whole kidney. Extracted RNA is then processed for downstream applications such as RNA-Seq and subsequent bioinformatical analyses. (B) Validation of macrophage-specific mRNA enrichment from MacTRAP kidneys. RT-qPCR analysis demonstrates strong enrichment of established macrophage marker genes in bound versus unbound kidney fractions (**p < 0.01); data are shown as mean ± SEM; n = 5.
