GSM6752620
Pregnant mice were injected with tribromoethanol at enough dose to keep alive but anesthetized. Then, the small fetuses were removed one by one. A small squash from the lung was used to identify the GFP+ pups at E12, using an EvosFLC cell imaging system (LifeTechnologiesTM, California, USA). Once they were identified, the metanephros area was removed under microscopy and harvested in ice cold dPBS. The tissue was then minced carefully with a razor blade and transferred to 1.7mL Eppendorf tube. 300 μL of TryPLETM Express (Gibco, New York, USA) was added and incubated at 37∘C for 5 minutes. Then, 600 μL of DMEM + 5% FBS was added to the Eppendorf tube. The mixture was homogenized by pipetting up and down. The solution was then filtered with a 40 μm nylon cell strainer. Tissue chunks were removed from the mesh surface, placed again in an Eppendorf tube and the process repeated a second time. Meanwhile, the flow-through filtrate was placed in a new Eppendorf tube and centrifuged at 4∘C, 150g for 5 minutes. The supernatant was removed and the pellet resuspended with resuspension buffer. All the tubes were combined in the end and the cells were ready for single-cell capture. For later timpoints, animals were anesthetized with tribromoethanol (300 mg/kg). P5 and P30 mice kidneys were excised and decapsulated. Then, the kidney cortices were dissected, minced with a razor blade, and transferred into a 15 mL tube with 5 mL of enzymatic solution (0.3% collagenase A [Millipore-Sigma], 0.25% trypsin [Millipore-Sigma], and 0.0021% DNase I [Millipore-Sigma]). The tubes were placed inside a shaking incubator (80 RPM) for 15 minutes at 37∘C. The solution was pipetted up/down 10 times with a sterile transfer pipette and allowed to settle for 2 minutes, and the supernatant was collected in a fresh tube on ice. The enzymatic solution was added to the 15 mL tube containing the remaining undigested cortices, and the digestion procedure was repeated a total of 3 times. The supernatants were pooled and centrifuged at 800 g for 4 minutes at 4∘C using a Sorvall RT7 refrigerated centrifuge (Sorvall, Newtown, CT). The cell pellet was resuspended in fresh buffer 1 (130 mM NaCl, 5 mM KCl, 2 mM CaCl2, 10 mM glucose, 20 mM sucrose, 10 mM HEPES, pH 7.4), and the suspension was poured through a sterile 100 μm nylon cell strainer (Corning Inc., Corning, NY) and washed with buffer 1. The flow-through was poured through a sterile 40 μm nylon cell strainer (Corning Inc.) and washed with buffer 1. The flow-through was centrifuged at 1,100 g for 4 min at 4∘C. The cell pellet was resuspended in 1.5 mL of resuspension buffer [PBS, 1% FBS, 1 mM EDTA, DNAase I (Millipore-Sigma)]. DAPI (Millipore-Sigma) was added to the cells to identify the living cells. The GFP positive cells were collected by Fluorescent-Activated Cell Sorting (FACS) and resuspended in DMEM (Dulbecco's Modified Eagle Medium, Gibco, Netherlands) with 10% FBS (Fetal Bovine Serum) for immediate use. The sorters were either an Influx Cell Sorter (Becton Dickinson, Franklin Lakes, NJ) or a FACS Aria Fusion Cell Sorter (Becton Dickinson), both located at the Flow Cytometry Core Facility at the University of Virginia. GFP-positive cells were washed in dPBS with 0.04% BSA twice, and the sorted GFP-positive cells were washed in dPBS with 0.04% BSA twice. The cells were counted with the CellometerMini (Nexcelom Bioscience, Massachussets, USA). Sorted cells with viability higher than 80% and absence of clumps were chosen to proceed. Experiments yielded less than 100,000 cells. Therefore, we used the protocol designed for Low Cell Input Nuclei Isolation. Briefly, we centrifuged the cells at 500g at 4∘C for 10 minutes, removed the supernatant carefully and resuspended the cells in a freshly prepared lysis buffer (1M Tris-HCl, 5M NaCl, 1M MgCl2, 10% BSA, 10% Tween-20). We optimized the lysis incubation time for 7 minutes on ice. Next, we immediately added freshly made washing buffer (1M Tris-HCl, 5M NaCl, 1M MgCl2, 10% Tween-20, 10% Nonidet P40, 5% digitonin and 10% BSA). This washing was performed twice, and then supernatant was removed for a final wash with diluted nuclei buffer (nuclei buffer 20X, 10X Genomics, diluted in nuclease free water to 1X). Once this was completed, we again removed the supernatant, resuspended the nuclei pellet and counted them with a Neubauer Chamber (Spencer, Buffalo, USA). In all our experiments we were able to target between 1000-5000 nuclei. Nuclei were loaded and captured using the Chromium System (10X Genomics, Pleasanton, CA) following the manufacturer's recommendation100 using the Chromium Next GEM (Cell-Gel Beads in Emulsion) Chip H with reagents of Chromium Next GEM Reagent Kits v1.1 (10X Genomics). Initially, nuclei were incubated with the transposition mix, that includes a transposase, and later the GEMs were barcoded and PCR amplified to generate the cDNA. The cDNA was then cleaned with Dynabeads and SPRIselect, end-repaired, adaptor-ligated and amplificated by PCR. The constructed libraries were sequenced on an Illumina HiSeq 2500/4000 platform (150-bp paired-end reads).