GSM7964070
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. RNA was extracted from TdTomato positive enriched sorted cells were processed following the manufacture instruction mRNA easy, Qiagen). Briefly, total RNA samples were assessed for concentration using a Nanodrop (Nanodrop 504 ND8000, Thermo Fisher Scientific, Carlsbad, CA) and quality using the TapeStation 505 (4200 TapeStation, Agilent Technologies, Santa Clara, CA). Up to 30 ng of total RNA per sample was used for poly-A mRNA selection. Libraries for RNA-Seq were prepared with Nugen Universal plus mRNA-Seq Kit (part number: 0508) to generate strand-specific RNA-seq libraries. The workflow consists of poly(A) RNA selection, RNA fragmentation and double-stranded cDNA generation using a mixture of random and oligo(dT) priming, followed by end repair to generate blunt ends, adaptor ligation, strand selection, and PCR amplification to produce the final library. Different index adaptors were used for multiplexing samples in one sequencing lane. The concentration of the amplified library was measured with a Qubit fluorometer and an aliquot of the library was resolved on a Bioanalyzer.