GSM8636212: WT-1, scRNA-seq; Mus musculus; RNA-Seq
Kidneys were harvested and minced into approximately 1 mm3 cubes. Enzymatic dissociation was performed in a 5 mL digestion buffer at 37 °C for 30 minutes. The digestion buffer contained 0.25 mg/mL of Liberase TM Research Grade enzyme (Roche, USA) and 50 µg/mL DNase I (New England Biolabs, USA). The reaction was deactivated by adding 10% fetal bovine serum (FBS). The resulting cell suspension was passed through a 70 μm cell strainer (Bioland, China), and cells were collected by centrifugation at 400 × g for 5 minutes at 4 °C. The cell pellet was treated with 3 mL red blood cell (RBC) lysis buffer (Invitrogen, USA) for 1 minute on ice, followed by another centrifugation at 400 × g for 5 minutes at 4 °C. Subsequent washes were performed with chilled 1x phosphate-buffered saline (PBS), and the cells were filtered through a 40 μm cell strainer (Bioland, China). The cells were then centrifuged at 400 × g at 4 °C for 5 minutes and resuspended in 500 µl pre-chilled 1x PBS with 2% BSA. Viability dye eBioscience™ Fixable Viability Dye eFluor™ 780 (ThermoFisher, #65-0865-14) was used to stain viable single cells for 15 minutes, followed by two washes with pre-chilled PBS. Viable single cells were sorted using a BD FACSAria Fusion flow cytometer (BD Biosciences) Single-cell suspensions with greater than 80% viability were submitted to Novogene Biotechnology Co., Ltd (Beijing, China). Each sample, consisting of approximately ten thousand cells, was loaded onto the Chromium Single Cell Controller (10X Genomics) for cell encapsulation into gel beads in emulsion (GEMs). Subsequent steps involved cDNA library construction using the Chromium Single Cell 3' Reagent Kits, GEMs breakdown, barcoded cDNA purification and amplification, fragmentation, A-tailing, adaptor ligation, and PCR amplification for sample indexing and scRNA-Seq library enrichment.