GSM9755275: Multiome, testis, WT2, ATAC; Mus musculus; ATAC-seq
Single cell suspensions from wild-type and Ago413-/- testis were obtained following the method described by Ascenção et al. Testis from 2-5 mice from the same genotype were collected, the tunica albuginea was removed, and tubules were placed in 10 ml of preheated (37°C) DMEM-F12 containing 2 mg of Collagenase 1A (Sigma) for 2 minutes with manual shaking. Digestion was stopped by two washes of the tubules with DMEM-F12. Next, the tubules were incubated in 10 ml of DMEM-F12 with 5 mg of trypsin (Sigma) and 7 mg/ml of DNAse I (Sigma) at 37°C with constant shaking (150 rpm) until the length of the tubules was about 1 mm (approximately 10 minutes). Digestion was stopped by addition of 3 ml of Fetal Bovine Serum (FBS, Sigma) and 30 ml of DMEM F-12. Digested tubules were strained on a 100 μm strainer and centrifuged at 15°C for 5 minutes at 600g. Before proceeding with nuclei isolation, spermatozoa were removed from the germ cell suspension samples by negative selection Magnetic Activated Cell Sorting (MACS) using an antibody against the acrosomal protein ACRV1 coupled with anti-rabbit IgG MicroBeads (Miltenyi Biotec) and following the protocol provided by the manufacturer. Briefly, cells were labeled with anti ACRV1 antibody (1:50 concentration) by incubation of 108 cells in MACS buffer (0.5% BSA, 2mM EDTA in PBS, pH7.2) for 15 minutes. Then, the labelled cells were incubated with anti-rabbit IgG MicroBeads for 20 minutes on ice and loaded into an LS Column (Miltenyi Biotec) previously rinsed with MACS buffer. After depletion of spermatozoa by retention in the column, cells were collected from the negative fraction by centrifugation at 1500rpm for 5 minutes at 4C and proceeded with nuclei isolation using 10X Genomics Demonstrated Protocol CG000365 Nuclei Isolation for Single Cell Multiome ATAC + Gene Expression Sequencing. Single cell RNA and ATAC sequencing libraries were prepared at the Cornell Genomics Facility using the 10X Genomics Chromium Next GEM Single Cell Multiome ATAC + Gene Expression kit.Nuclei were processed on the 10X Genomics Chromium System, targeting a total of 10000 cells per sample. Quality control was evaluated using a Fragment Analyzer