GSM4790066: adult mouse pituitary 1; Mus musculus; ATAC-seq
Pituitaries were collected from two adult male mice. Nuclei isolation for scATAC-seq was performed following the instruction provided by 10xGenomics (scATAC-V1) with some modifications. 100,000 cells were added to a 2 mL microcentrifuge tube and centrifuged (300 x g for 5 min at 4°C). The supernatant was removed without disrupting the cell pellet. Lysis Buffer [10 mM Tris-HCl (pH 7.4), 10 mM NaCl, 3 mM MgCl2, 0.1% Tween-20, 0.1% Nonidet P40 Substitute, 0.01% Digitonin, and 1% BSA] was diluted by Lysis Dilution Buffer [10 mM Tris-HCl (pH 7.4), 10 mM NaCl, 3 mM MgCl2, 1% BSA] to make 0.1x Lysis Buffer. 100 μL chilled 0.1x Lysis Buffer was added to the cell pellet, and then pipette-mixed 10 times. The microcentrifuge tube was then incubated on ice for 5 min. Next, 1 mL of chilled Washing Buffer [10 mM Tris-HCl (pH 7.4), 10 mM NaCl, 3 mM MgCl2, 0.1% Tween-20, and 1% BSA] was added and gently pipette-mixed 5 times. Nuclei were centrifuged (500 x g for 5 min at 4°C) to remove the supernatant, and the wash was repeated. An appropriate volume of chilled Diluted Nuclei Buffer (10xGenomics) was added to resuspend nuclei at 2,500 nuclei/μL to retrieve/target 6,000 nuclei per library following the standard protocol provided by Chromium Single Cell ATAC Library and Gel Bead kit (10x Genomics, v1). Libraries were constructed using standard protocol provided by Chromium Single Cell ATAC Library and Gel Bead kit (10x Genomics, v1). Each sample was uniquely barcoded Libraries were then pooled and loaded on a NovaSeq Illumina sequencer and sequenced to ~50% saturation