Spatial transcriptomics analysis of mineralized tissues faces significant challenges due to lengthy decalcification procedures that severely compromise RNA integrity and subsequent gene detection.This protocol details...Spatial transcriptomics analysis of mineralized tissues faces significant challenges due to lengthy decalcification procedures that severely compromise RNA integrity and subsequent gene detection.This protocol details an optimized workflow to process bone and fracture callus samples for 10x Genomics Visium spatial transcriptomics.The key innovation involves replacing conventional EDTA decalcification with Morse's solution,enabling rapid decalcification(<24 h)while preserving RNA quality,as evidenced by a favorable DV200 value.Additionally,the protocol emphasizes the critical use of SCHOTT NEXTERION®Slide H(3-D hydrogel-coated)to maximize adherence of fragile decalcified bone sections during processing,preventing detachment and preserving morphological integrity.We applied this optimized method to intact and fractured mouse femurs.The results demonstrate a substantial improvement in transcript capture efficiency:Visium V2 yielded an average of 5639 genes per spot,while Visium HD achieved an average of 170 genes per 8μm bin(equivalent to~4100 genes per 55μm spot).This step-by-step protocol overcomes major pre-analytical hurdles,enabling highresolution spatial transcriptomic profiling of mineralized tissues with significantly enhanced data quality.展开更多
基金funded by the National Key R&D Program of China(No.2021YFA1102600)the National Natural Science Foundation of China(Nos.82103781,and 82472433)the Science Fund for Distinguished Young Scholars of Liaoning Province(No.2024JH3/10200034).
文摘Spatial transcriptomics analysis of mineralized tissues faces significant challenges due to lengthy decalcification procedures that severely compromise RNA integrity and subsequent gene detection.This protocol details an optimized workflow to process bone and fracture callus samples for 10x Genomics Visium spatial transcriptomics.The key innovation involves replacing conventional EDTA decalcification with Morse's solution,enabling rapid decalcification(<24 h)while preserving RNA quality,as evidenced by a favorable DV200 value.Additionally,the protocol emphasizes the critical use of SCHOTT NEXTERION®Slide H(3-D hydrogel-coated)to maximize adherence of fragile decalcified bone sections during processing,preventing detachment and preserving morphological integrity.We applied this optimized method to intact and fractured mouse femurs.The results demonstrate a substantial improvement in transcript capture efficiency:Visium V2 yielded an average of 5639 genes per spot,while Visium HD achieved an average of 170 genes per 8μm bin(equivalent to~4100 genes per 55μm spot).This step-by-step protocol overcomes major pre-analytical hurdles,enabling highresolution spatial transcriptomic profiling of mineralized tissues with significantly enhanced data quality.
