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The rapid development of single-cell sequencing technology offers new opportunities for studying the cellular heterogeneity and functional diversity of adipose tissue. However, a series of preprocessing steps is required before single-cell sequencing of rabbit adipose tissue to ensure the accuracy and reliability of the sequencing data.
This article will focus on the key preprocessing steps for single-cell sequencing of rabbit adipose tissue, including the acquisition of single-cell suspension, cell separation, cell capture, and RNA extraction. Proper execution of these steps will provide high-quality samples and data for subsequent single-cell sequencing experiments, offering strong support for the research on rabbit adipose tissue.
Material Preparation
● Rabbits
● RWD High Activity Adipose Tissue Enzymatic Digestion Kit（Mouse and Rat）
● RWD Single Cell Suspension Dissociator
The following are the simplified steps
Take the adipose tissue and repeatedly rinse it with PBS until clean. Then store the tissue temporarily in a container with Hanks buffer (containing Ca2+ and Mg2+) or RPMI 1640. Cut the tissue into 2 ~ 4 mm pieces. Weigh the pieces for targeted weight using an electronic balance.
The adipose tissue should be taken upon immediate use. Lymph nodes, blood stains, and redundant connective tissue septa that may be contained in each adipose tissue should be removed as much as possible.
Transfer the pieces of adipose tissue into a tissue processing tube containing enzyme mix.
Run the program M-Adipose-Heater-1 on the single-cell suspension dissociator.
Rinse the 100 μm cell strainer and filter the cell suspension sample using the wetted 100 μm cell strainer. Collect the cell suspension into a 50 mL centrifuge tube.
The results counting cells by the C100 counter after obtaining the cell suspension, showed that traditional grinding methods produced many cell debris while foaming, as if grinding soy milk. After switching to the RWD Single Cell Suspension Dissociator and the enzymatic digestion kit, the number of cell debris decreased, and cell viability increased.
