Quality control measurements demonstrated that these optimizations eliminated cellular debris and allowed for a high yield of high-quality nuclei and RNA for library preparation and sequencing. Using snRNA-seq, 16 distinct kidney cell clusters were recovered from normal and peri-transplant acute kidney injury allograft samples, including immune cell clusters. This method is effective for obtaining normal nuclei morphology without signs of structural damage. Samples can be processed in 90 min or less. The described protocol is fast, low cost, and time effective due to the elimination of cell sorting and ultra-centrifugation. Here, an optimized protocol for single nuclei isolation is presented for frozen and RNA later preserved human kidney biopsies. However, the study of complex tissues using small core biopsies presents many technical challenges. snRNA-seq largely relies on the dissociation of intact nuclei from human tissues. Single cell resolution enables the study of novel cell types, biological processes, cell trajectories, and cell–cell signaling pathways. (11)01108-5/fulltext?referrer=https%3A%2F%.Single nuclei RNA sequencing (snRNA-seq) has evolved as a powerful tool to study complex human diseases.Mammospheres from single cells in Matrigel®.ĥe 3 lung epithelial cells + 5e 4 lung mesenchymal cellsĭissociated crypt cells or hESC in Matrigel®. Table: Examples of various organoid cultures, culture conditions, and commonly used enzymes for organoid dissociation.ĭissociated mammary gland to obtain organoids. It is important to note that the cells from organoid cultures could potentially show different gene expression profiles than those observed from primary tissues.Wash steps should be performed to remove any unwanted debris. Cell viability should be higher than 90% post-organoid dissociation step in order to obtain the best results for single-cell analysis.Optimization of enzyme digestion time should be considered in order to obtain healthy single cells during dissociation steps. Refer to the table below for the use of appropriate enzymes based on the organoid type. After matrix dissociation, preparation of single cells from organoids could be achieved by using different enzymes such as Accutase ®, Papain, Trypsin, TrypLE ™ or Dispase.In order to obtain healthy cells for the analysis, it is important to minimize incubation time during 3D matrix dissociation. Prior to processing organoids for single-cell analysis, the 3D culture should be dissociated using reagents such as Dispase or Collagenase. Typically, organoids are placed in 3D cultures using Matrigel ® or collagen.High seeding density could result in undesirable outcomes such as smaller organoids and potential cell death. Avoid overcrowding of the organoid cultures. Each organoid culture is limited by the surface area of the culture dish. It is important to consider the seeding density of cells at the start of the organoid culture.Experimental procedures should be optimized to minimize unnecessary prolonged tissue handling and enzymatic digestion during tissue association. ![]() If organoids are prepared from adult stem cells it is important to take proper care in preparing a single-cell suspension from the tissue of origin.A pilot study is recommended before starting a large-scale experiment. Note that we have not specifically tested these approaches, they are meant to be general guidelines. Below, you will find a schematic diagram of an ideal workflow along with some of our recommendations for working with organoid tissue as input to 10x Genomics Single-Cell Assays. For example, organoid cultures could be generated using iPSCs, hESCs, or adult stem cells. Organoids are generated using relevant cell culture strategies based on the cells of origin. These 3D cultures provide useful information regarding the biology of healthy or diseased organ models. Question: Are there any recommendations for working with organoid tissue for 10x single-cell assays?Īnswer: Organoids are 3-dimensional (3D) cell cultures that represent fundamental characteristics and cellular organization of an organ.
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