spatial transcriptome sequencing of normal developing ovule and abortive ovule
The tissue samples was obtained from healthly donors and patients or mouseand immediately remove the types of tissue not needed for the study, such as connective tissue and adipose tissue. And tissue surfaces were quickly rinsed with a pre-cooled solution of 1X PBS (RNase free) or normal saline to remove residual blood, and sterile gauze was used to blot the surface fluid. The tissue size was required to be suitable, and the tissue should be cut into small pieces(6.8mm2) suitable for subsequent experiments. Small fragments of each tissue were snap-frozen in isopentane pre-chilled with liquid nitrogen and optimum cutting temperature (OCT) compound (SAKURA,Cat#: 4583) and stored at 80 until use.The Spatial Transcriptomics (ST) protocol was optimized for tissue according to recommendations (Salmen et al., 2018b). In short, changes were made in the staining procedure by excluding isopropanol, decreasing the incubation time of hematoxylin and bluing buffer, as well as increasing eosin concentration. Moreover, the optimal incubation time for permeabilization was established, and the previously described one-step protocol for tissue removal was altered by using a higher proteinase K:PKD buffer ratio. Once optimal conditions had been established, three cryosections per patient were cut at 10 mm thickness onto spatial slides and processed immediately.