GSM9015195: kidney, cholesterol crystal embolism, scRNA (SCX-CCE-14); Mus musculus; RNA-Seq
After the kidneys are excised, wash the surface blood with saline, HBSS, or PBS, and place them in a sterile culture dish. Wash three times with HBSS balanced salt solution, then transfer into a DNFU dissociation bottle. Add an appropriate amount of tissue dissociation solution and set the tissue dissociation instrument program to 180rpm-36 cycles-15 minutes. Run the program once and observe under a microscope to check if the kidney tissue is fully dissociated. If not, increase the number of cycles as needed to obtain a single-cell suspension. After digestion, filter the cell suspension using a 40μm filter, and use PBS with 0.04% BSA as the buffer solution. Centrifuge the filtered cell suspension at 350×g for 5 minutes, discard the supernatant, and resuspend the cell pellet in PBS. Then, add red blood cell lysis buffer at a 1:2 volume ratio to the cell suspension and incubate on ice for 5-8 minutes. Centrifuge at 300×g for 5 minutes, discard the supernatant, and resuspend the cell pellet in 1mL PBS. Wash again by adding PBS to bring the volume to a constant level, centrifuge at 300×g for 5 minutes, discard the supernatant, and resuspend the cell pellet in an appropriate amount of PBS. We used GEXSCOPETM Single Cell RNA Library Kit Tissue (Singleron Biotechnologies) to barcode single cells, capture mRNA from isolated single cells, and generate cDNA libraries for scRNA-seq. Then, individual libraries were diluted to 4 ng/μl, and pooled for sequencing on a HiSeq 4000 platform (Illumina, San Diego, USA) with 150 bp paired-end reads.