GSM8995847: Kidney, old, male, SGLT2; Nothobranchius furzeri; RNA-Seq
We established and verified our own single nuclei RNA isolation protocol for killifish kidneys. Our protocol was established by combining two existing protocols, one for Killifish brain (Teefy et al., 2022) and a kidney-specific protocol for mice (Leiz et al., 2021). For the experiment the following numbers of fish were used: young female n=9, young males n=6, old females n=6, old males n=5. Pooling of multiple kidneys was necessary to achieve an average tissue weight of 20 mg. Young time points were defined as 6 weeks old animals; old time point was defined by the median lifespan of the cohort (in this case: 20 weeks of age). All old fish were transcardially perfused with ice-cold PBS before kidney collection (Mariën et al., 2022). Kidneys were pooled into RNA-later solution and stored at 4°C for 24h. Then all remaining liquid was removed, and tissue was moved to -80°C until isolation. Nuclei isolation: On day of experimentation tissue was removed from the cryotube, rehydrated with ice-cold PBS on ice. PBS was removed and tissue was resuspended in Nuclei Lysis Buffer (NLB: 10mM Tris-HCl (pH 7.4), 10mM, NaCl, 3mM MgCl2, 0.01875% v/v NP-40, 0.2U/µl RNase Inhibitor). Tissue in buffer was transferred into a 2ml dounce homogenizer on ice and homogenized with pestle A for 30 sec. The homogenate was passed through 70µM strainer into a 15 ml tube, previously precoated with 5% BSA overnight. The filter was washed with another 1ml of NLB. The dissociated tissue was moved back into the douncer and homogenized using pestle B for 1 minute. The homogenate was then strained through a 40µm strainer into a 15 tube precoated with 5% BSA. The homogenate was diluted with Nuclei wash buffer (NWB: 2% BSA in PBS, 0.2U/µl RNase Inhibitor) up to 5 ml and gently inverted. The suspension was centrifuged at 500g at 4°C for 10 minutes. Nuclei purification: Nuclei were purified with an additional wash step. The supernatant was discarded by gently tipping over the tube and the pellet was resuspended in 1 ml NWB using a wide bore P1000. The volume was filled up to 5 ml with NWB and the tube centrifuged 500g at 4°C for 5 minutes. Debris removal: The supernatant was carefully discarded by gently tipping over the tube. The pellet was resuspended in 1 ml NWB with a wide bore P1000. 300µl debris removal solution (Miltenyi Biotec®, USA) was added and slowly mixed. The solution was overlayed with 1ml NWB and centrifuged at 3000g for 10 minutes at 4°C. The top two phases were gently removed. The remaining phase was diluted to 8ml and centrifuged at 1000g for 10 minutes at 4°C.  The supernatant was removed and the pellet resuspended in 1 ml NWB. The solution was filtered through a 20µm filter into a 1.5ml low-bind DNA tube. Samples were processed which have shown an RNA Integrity number of >7 as measured on a Agilent Bioanalyzer using a Bioanalyzer High Sensitivity RNA Analysis Kit (Agilent®, USA). Nuclei were counted using Acridine Orange/Propidium Iodide Assay on a CellDrop®( DeNovix®, USA) automated cell counter set to “Nuclei AO/PI”. A Chromium Next GEM Single Cell 3' Reagent Kit v3.1 (10x genomics) was used for library construction following the user's manual. Nuclei were concentrated to 1000nuclei/µl for a targeted cell recovery of 7000.