GSM9465318: snATAC-seq of nuclei isolated from cropreserved endosteal cells; Mus musculus; ATAC-seq
Endosteal cells were isolated from femora and tibiae of 9-week-old male C57BL/6J mice as described above. Single-cell suspensions were washed with PBS containing 1% BSA and pelleted (500 × g, 5 min, 4 °C). Cells were lysed in 100 µL custom lysis buffer (0.0075% digitonin, 0.05 mg/mL protease inhibitor [Millipore Sigma, 114298668001], 20 mM Tris-HCl [pH 7.4], 150 mM NaCl, 3 mM MgCl₂, 2% BSA) on ice for 1–2 min with gentle pipetting to facilitate lysis. Nuclei were washed with washing buffer (0.025 mg/mL protease inhibitor, 20 mM Tris-HCl [pH 7.4], 150 mM NaCl, 3 mM MgCl₂, 1% BSA) and pelleted (300 × g, 8 min, 4°C). Single-nucleus ATAC-seq (snATAC-seq) was performed using the 10x Genomics Chromium Single Cell ATAC v2 platform, following the manufacturer's protocol. Briefly, nuclei were counted using a LUNA counter and haemocytometer to assess concentration and nuclear integrity. Nuclei were diluted to the recommended loading concentration and transposed using the Tn5 transposase in the ATAC transposition mix. Following transposition, gel bead-in-emulsions (GEMs) were generated using the Chromium Controller, enabling barcoding of accessible chromatin fragments in individual nuclei. Post-GEM amplification and library construction were carried out according to the 10x Genomics protocol. Final libraries were quantified using Qubit and assessed for fragment distribution using the Agilent Bioanalyzer or Tapestation.