GSM8006798: GPAD-20220124-NMK92M1(snATAC,male,week 92); Mus musculus; OTHER
C57BL/6J mice were originally purchased from the Jackson Laboratory. Mouse kidneys were collected at embryos (E16.5), newborns (P0), 3 weeks (W3), 12 weeks (W12), 52 weeks (W52), and 92 weeks (W92). To get E16.5 kidneys, 4 female mice and 1 male mouse were grouped for mating on day 1 at 5 pm. Then the male was removed from the females on day 2 at 10 am. Day 2 was designated as E0.5 for any pregnant females. For kidneys collected from other ages, mice were purchased from the Jackson Laboratory. Mice at E16.5 and P0 were euthanized by decapitation. Older mice were euthanized by carbon dioxide asphyxiation. For each mouse, one kidney was embedded in optimal cutting temperature compound (OCT) and the other one was fixed in 10% neutral buffered formalin (Epredia, 5725) then embedded in paraffin. For Visium ST, 10 μm mid-sagittal OCT sections were used on the 10x Genomics Visium platform; for snRNA-Seq and snATAC-Seq, pooled snap-frozen tissues were used for E16.5 and P0 mice, and 300 μm of mid-sagittal OCT sections were used for mice from W3 to W92. All animal experiments were approved by the Washington University in Saint Louis Institutional Animal Care and Use Committee (IACUC) office. Nuclei lysis buffer was freshly prepared in cold nuclease-free water with a mixture of 10 mM Tris-HCl (pH=7.4) (Thermo; 15567027), 10 mM NaCl (Thermo; AM9759), 3 mM MgCl2 (Thermo; AM9530G), 0.01% NP40 substitute (Sigma, 74385-1L), 1% BSA (MACS, 130-091-376), 1 mM DTT (Sigma, 646563) and 0.4 U/μL Protector RNase inhibitor (Sigma, 3335402001). Cryopreserved tissues were lysed with the cold lysis buffer in the Dounce homogenizer (KONTES, 885300-0002) using pestle B. The homogenate lysate was then filtered through a 40 μm strainer and washed with the wash buffer (2%BSA + 1 x PBS + 0.4 U/μL Protector RNase inhibitor). The filtrate was centrifuged at 500g for 6 minutes at 4°C and resuspended in 500 μL wash buffer for fluorescence-activated cell sorting (FACS). Nuclei were stained with 7-AAD (7-Aminoactinomycin D) dye (Millipore Sigma SML1633-1ML) at a 1:200 dilution. The suspension was FACS sorted for 400,000 positively stained nuclei. After sorting the nuclei suspension was centrifuged at 500g for 6 minutes at 4°C, the pellet was resuspended in 2x Nuclei Dilution Buffer (10x Genomics, 2000153) supplemented with 1 M DTT and 1.2 U/μL Protector RNase inhibitor. Nuclei were stained with Trypan blue 0.4% (Invitrogen, T10282) and quantified using a hemocytometer, 20,000 nuclei were transposed and partitioned into Gel Beads-in-emulsion (GEMs) in the Chromium Controller (10x Genomics, Pleasanton, CA) following Chromium Next GEM Single Cell Multiome ATAC + Gene Expression protocol CG000338 Rev A (10x Genomics). The transposed DNA and cDNA of each individual nucleus were separately indexed and then made into libraries following the protocol. All libraries were sequenced on Illumina NovaSeq 6000 system with specific flow cell types (snRNA-Seq: S4; snATAC-Seq: S1). Measurements related to snRNA-Seq and snATAC-Seq were taken from distinct samples.