The glomerular circadian clock temporally regulates basement membrane dynamics and glucocorticoid response
The glomerular circadian clock temporally regulates basement membrane dynamics and glucocorticoid response
The care and use of all mice in this study was carried out in accordance with the UK Home Office regulations, UK Animals (Scientific Procedures) Act of 1986 under the Home Office Project Licences: P1AE9A736, PP4262564 and PP7202624. Mice were maintained at 20°C to 22°C, humidity of 40-50%, with ad libitum access to water and standard rodent chow. Wild type C57/BL6J mice housed in 12-h light/12-h dark cycle (LD) were used for time-series cell harvesting. 8-week-old male wildtype (C57BL/6) mice were kept in DD 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 LD schedule; CT0 indicates 6am. Kidneys were harvested at 4 h intervals for 48 h and immediately snap frozen and stored at -80oC for later processing. Two biological replicates were obtained at each time-point. Glomeruli were sieve-isolated from thawed kidneys. Kidneys were cut into 1-2mm3 pieces and passed through a 100 μm cell strainer (Endecotts, UK) and then flow-through was passed through a 70 μm cell strainer (Endecotts, UK). For the isolation of RNA from glomeruli, glomeruli were re-suspended in 1ml of TRIzol (Thermo Fisher Scientific) and kept at RT for 5 mins prior to the addition of 175μl of chloroform. This sample was shaken vigorously and kept at RT for 3 mins before being centrifuged at 4oC for 10 mins at 13,000 rpm. The aqueous phase was removed by aspiration and transferred to a sterile RNase-free tube with 500μl of isopropanol. After 15 minutes at RT, the sample was further centrifuged at 4oC for 10 mins at 13,000 rpm and the supernatant was carefully discarded. After drying by evaporation, 1ml of 75% RNA-free ethanol was added and the sample was further centrifuged at 4oC for 10 mins at 13,000 rpm. The supernatant was carefully discarded, and the sample was allowed to dry by evaporation for 10 mins. Following this, the pellet was re-suspended in 40μl of nuclease-free water and incubated at 65oC for 5 mins. The quality of resuspended mRNA was then measured using a QubitTM Fluorometer (Thermo Fisher Scientific) and RNA integrity measured using a 2200 TapeStation (Agilent Technologies) to ensure good quality for downstream analysis. Illumina stranded mRNA library