Our method helps to generate and understand gene expression data from complex tissues on a single-cell level. Our method can be applied to basically any tissue in the same manner and leads to high-quality data with very little protocol-related artifacts. Dr.Janna Leiz and myself from the Schmidt-Ott lab will be presenting the protocol.
Begin by placing the kidney on a cold dissecting plate and use a sharp scalpel or razor blade to obtain a middle slice of one to two millimeters. Make sure the tissue piece contains the entire corticomedullary axis. Use microdissection scissors and forceps to carefully trim the cortex from the sides of the center piece.
Within the dissected tissue piece, the three segments cortex, outer medulla, and inner medulla should be clearly visible. Transfer the kidney piece to the previously-prepared RNA stabilization solution and incubate for 24 hours at four degrees Celsius to avoid RNA degradation. After 24 hours, remove the RNA stabilization solution.
Carefully remove the excess solution with a tissue paper and store the tissue at minus 80 degrees Celsius until further use. Take the frozen kidney piece and transfer it to a 60-millimeter, pre-cooled polystyrene Petri dish on ice containing one milliliter of Nuclei Lysis Buffer 1, or NLB1. Mince the tissues thoroughly using a razor blade or scalpel.
Cut off the tip of a one-milliliter pipette tip and transfer the minced tissue and buffer to the grinder tube placed on ice. Ensuring that all the pieces have been transferred, wash the Petri dish five to 10 times with the buffer. Slowly move the pestle A 25 times up and down in the grinder tube on ice to homogenize the suspension.
Pass the homogenate through a 100-micrometer strainer in a pre-cooled 15-milliliter collection tube on ice and wash the filter with another one milliliter of NLB1. Wash the grinder tube with cold EZ nuclei lysis buffer and discard the buffer. Transfer the homogenate back into the grinder tube and slowly move the pestle B 15 times up and down in the grinder tube on ice to homogenize the suspension.
Transfer the homogenate to a pre-cooled 15 milliliter collection tube on ice. Wash the grinder tube with another two milliliters of NLB1, and make sure to transfer all tissue fragments to the collection tube. Incubate the homogenate for five minutes on ice to lyse the cells.
Pass the homogenate through a 40-micrometer strainer into a pre-cooled 15-milliliter collection tube. Spin the collection tube for five minutes at 500 times G at four degrees Celsius in a centrifuge with a swinging-bucket rotor. In the meantime, add RNase inhibitor solution to NLB2.
Remove the supernatant without disturbing the pellet. Carefully resuspend the pellet in four milliliters of NLB2. Carefully underlay the suspension with a one-milliliter cushion of sucrose gradient buffer.
Spin the suspension at 500 times G for five minutes at four degrees Celsius in a centrifuge with a swinging-bucket rotor. Meanwhile, add RNase inhibitor solution to the nuclei suspension buffer. After centrifugation, gently remove the collection tube from the centrifuge, being careful not to disturb the two layers when handling the collection tube.
The cell debris can be observed between the two layers. Remove the supernatant carefully, starting with the debris. Remove the remaining supernatant without disturbing the nuclei pellet and carefully resuspend the pellet in one milliliter of nuclei suspension buffer.
Pass the homogenate through a 20-micrometer strainer into the pre-cooled, five-milliliter fluorescence-activated cell sorting collection tube. The number of genes was plotted against the number of transcripts defined by unique molecular identifiers and colored by the fraction of mitochondrial reads to assess the quality of the nuclei isolated. A total of 20, 000 genes were detected in 6, 000 nuclei with 1, 600 median genes and 2, 800 median unique molecular identifiers per nucleus.
Gene expression patterns of cluster-enriched markers were visualized using a dot plot, and cell type clusters in a t-distributed stochastic neighbor embedding plot. The percentage of each cell type was calculated and used to determine the ratio of proximal tubule to thick ascending limb cells. It is critical to work at four degrees Celsius at all times and to determine the optimal amount of tissue in trial runs to ensure high-quality suspensions and optimal concentrations.
