GSM7964041
Briefly cortices and outer medulla regions of the kidney were dissected and thoroughly minced in 1mlcold DMEM/F12 media using sterile fine forceps. The minced tissue  resuspended in fresh 1ml DMEM/F12 media supplemented with 7.5mg/ml of B. Licheniformis Cold Active Protease, 125U/ml DNase1  and 0.1mg/ml Liberase TL  and continuously kept on ice for 1 hour with frequent pipetting using 1ml pipette every 10 min. After complete dissociation of tissues into single cells (an aliquot of cell suspension was taken every 20 min to check dissociation of the tissue under the microscope), the enzymatic reaction was stopped using cold DMEMF12 media + 10% FBS. The cell suspension was filtered through a 40μm cell strainer. The filtrate was then centrifuged at 300g for 5 min and transferred to FACS tubes (BD Biosciences). Cells were washed in PBS and resuspended in 1.5ml of FACS sorting buffer containing 1mg/ml DAPI (1:1000, Life Technologies). Cell sorting was performed using the FACSAria III cell sorter (BD Biosciences). Cell populations were gated to exclude debris and doublets using FACSDiva software (BD Biosciences). Cells were collected from relevant gated cell populations in 1.5mL collection tubes (Eppendorf) containing collection buffer. Cells pellets of around 100.000 cells from 2 or 10 days post injury were lighly fixed in 0.2% PFA and freeze at –80 for storage until all samples were collected. Each sample was split in 3 aliquots for subsequent processing with  Sox9 antibody (#AB5535, Millipore), as a positive control with H3K4me3 (#9751, Cell signaling) or as an input control. CUT&RUN assays were carried out with the Cell Signalling assay kit and protocol (Catalog number #86652, Cell Signaling) and next-generation sequencing libraries were generated from the immuno-enriched DNA samples using the DNA Library Prep Kit for Illumina with the Multiplex Oligos for Illumina Systems (Dual Index Primers) kits (#56795 and #47538, Cell Signaling, respectively). The final libraries quality was assessed by fragment size in a TapeStation before sequencing.