title: GSM8717485: Non-leukocyte kidney cells of low potassium diet; Mus musculus; RNA-Seq
library_construction_protocol: To obtain non-leukocyte kidney cells, whole kidneys of mice were minced and incubated within the collagenase P solution described in the flow cytometry section. The tissue chunks were further digested by pipetting and passing through thin syringe needles multiple times. After enriching the cells using the Percoll gradient method, cells were incubated with Hoechst33342 at 37 °C for 15 min and then labeled with PE/Cy7-conjugated anti-CD45 antibody. Hoechst33342+CD45–Sytox– cells, as live non-leukocytes, were sorted using FACSAria II (BD). Cells were resuspended in Cell Banker 1 (Zenogen Pharma, Koriyama, Japan) and stored at -80 °C until ready for the gel bead emulsion process. Samples with high viability were utilized, yielding either normal K+ (N) or low-K+ (L) (n=2 for each), and were processed using a dead cell removal kit (Miltenyi, Bergisch Gladbach, Germany). Cells were processed using the 10x Genomics Chromium controller and Chromium Next GEM Single Cell 3ʹ Reagent Kits v3.1 following manufacturer's instructions. Each treatment group cell mixture (N or L, n=1 for each) was loaded, aiming for a targeted cell recovery of 6,000 cells. The library was prepared using 10xGenomics's Chromium Next GEM Single Cell 3ʹ Reagent Kits v3.1 and dual index kit following manufacturer's instructions.  Illumina P5 and P7 sequences and sample index sequences are added during the Sample Index PCR. The final library fragments contain the P5, P7, Read 1 and Read 2 sequences used in Illumina bridge amplification and sequencing. Additionally, each fragment contains the 10x Barcode, UMI and cDNA insert sequence used in data analysis.  Sequencing was conducted by Azenta (Leipzig, Germany) using a NovaSeq 6000 (Illumina, San Diego, CA, USA).