GSM7964191
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. TdTomato positive enriched sorted cells were processed following the manufacture instruction. Sorted cells nuclei were isolated as described in 10xGenomics protocol (CG000169 Nuclei Isolation for Single Cell ATAC Sequencing), following the low input version and with a 1:5 diluted Lysis Buffer in Nuclease-free water. Isolated cell samples were immediately processed with a Chromium Next GEM Single Cell ATAC Kit v2 (10xGenomics) snATAC-seq