GSM7653941: S858R Kidney 2 snRNA-seq; Mus musculus; RNA-Seq
We adapted and conducted single nuclei isolation for the kidney cortex as previously described by Kirita et al. PNAS 2020. We isolated nuclei using the Nuclei EZ Prep kit with Nuclei EZ Lysis buffer (Sigma #NUC-101) supplemented with cOmplete ULTRA tablets (Sigma# 05892791001), SUPERase IN (Thermosisher# AM2696), and Promega RNasin Plus (Fisher Promega #PRN2615). We minced whole kidney cortex into <1mm pieces and then homogenized them using a Dounce tissue grinder (Kimble# 8853000002) in 2 ml of cold Nuclei EZ Lysis buffer. We then filtered the homogenate through a 200-µm strainer (Pluriselect#:43-50200) and a 40-µm strainer (Pluriselect#:43-50040) and then centrifuged at 500 x g for 5 min at 4 ºC. We resuspended the pellet in 4ml of the buffer and incubated it on ice for 5 minutes. After centrifugation at the same time, temperature, and speed, we resuspended the pellet in Nuclei Suspension Buffer as described by Kirita et al. PNAS 2020.  We adapted our single nuclei isolation for cerebral cortex samples for mice from the previously described protocol for rats in Phillips et al. Cell Reports 2022 and sorted nuclei at the University of Alabama at Birmingham's (UAB) Comprehensive Flow Cytometry Core (CFCC) using fluorescence-activated cell sorting (FACS) on an Atlas BD ARIA II. Per the standard 10X protocol, the UAB CFCC used the 10X Chromium Controller to partition kidney and cerebral cortex nuclei into droplets with a barcoded gel bead where nuclei were lysed, and RNA was reverse transcribed, creating cDNA encapsulated in each droplet. After fragmenting and amplifying cDNA, they added Illumina adapters using the Chromium Single Cell 3ʹ GEM, Library & Gel Bead Kit v3 (10X#: PN-1000121). The UAB Heflin Center for Genomic Sciences at the UAB Sequencing Core indexed and sequenced all samples on a NovaSeq 6000 (Illumina). For each sample, they loaded approximately 20,000 nuclei onto an S4 flow cell.