Glomerular circadian transcriptomics (meta2d BHQ <0.1)Podocyte circadian transcriptomics (meta2d BHQ <0.1)Kidney circadian proteomics meta2d (p val <0.05)

For circadian kidney collection, eight-week-old male and female wildtype (C57BL/6J) mice were kept in Dark-Dark for 36 h prior to the onset of tissue collection and until they were sacrificed by cervical neck dislocation. Circadian time (CT) corresponds to administration of light in the animal room in normal Light-Dark schedule; CT0 indicates 6am. Kidneys were harvested at 4 h intervals for 48 h and immediately snap frozen and stored at -80^oC for later processing. For the glomerular circadian transcriptomics, glomeruli were sieve-isolated from thawed kidneys and RNA was isolated using TRIzol. RNA was checked for quality and prepared for sequencing in the Genomic Technologies Core Facility in Manchester. RNA-seq counts were normalized using DESeq2 (v1.28.1) and rhythmic genes were identified with MetaCycle. For the kidney circadian proteomics, kidneys from eight-week-old male and female wildtype (C57BL/6J) mice harvested during the time-series tissue collection described above were used for mass spectrometry-based proteomics. Mouse kidneys were enriched for matrix proteins and peptide samples were analyzed by liquid chromatography (LC)-tandem mass spectrometry using a Thermo Exploris 480 mass spectrometer (Thermo Fisher Scientific). The meta2d function from Metacycle was used to identify rhythmic proteins detected in at least 2 out of the 3 replicates per circadian time.For the time-series cell collection, primary podocytes were cultured from kidneys of wildtype mice (C57BL/6J, aged eight weeks) for 14 days. Podocytes were harvested in BL buffer containing 0.1% 1-Thioglycerol (Promega) and immediately snap-frozen at 4 h intervals for 48 h starting at 15 h post-synchronization with dexamethasone. RNA was isolated and purified using a ReliaPrep RNA Cell Miniprep System (Promega) as per manufacturers protocol. RNA was checked for quality and prepared for sequencing in the Genomic Technologies Core Facility in Manchester. RNA-seq counts were normalized using DESeq2 (v1.28.1) and rhythmic genes were identified with MetaCycle.