GSM8139430: pediatric hMSCs, undifferentiated, 16 year-old, male, donor 5; Homo sapiens; ATAC-seq
Fresh hFOBs, pediatric osteoblasts, and osteoclasts were harvested via Trypsin or TrypLE, followed by a series of DPBS washes steps. 50,000 cells from each sample were pelleted at 550 × g for 5 minutes at 4 °C. The cell pellet was then resuspended in 50 μl cold lysis buffer (10 mM Tris-HCl, pH 7.4, 10 mM NaCl, 3 mM MgCl2, 0.1% IGEPAL CA-630) and centrifuged immediately at 550 × g for 10 minminutes at 4 °C. The nuclei were resuspended in transposition reaction mix (2x TD Buffer (Illumina Cat #FC-121–1030, Nextera), 2.5 µl Tn5 Transposase (Illumina, 20034197 Cat #FC-121–1030, Nextera) and Nuclease Free H2O) on ice and then incubated for 45 minminutes at 37°C. The transposed DNA was then purified using the MinElute Kit (Qiagen), eluted with 10.5 μl elution buffer (EB), frozen and sent to the Center for Spatial and Functional Genomics at CHOP. Chondrocytes were processed using a similar procedure which involved taking from each human donor (N=3), suspensions of 75,000 cells. These were centrifuged at 550 X g for 5 min at 4°C, resuspended in 50 µl of ice-cold PBS, and centrifuged once more at 550 X g for 5 minutes at 4 °C. The final cell pellets were resuspended in 50 µl of ice-cold lysis buffer (10 mM Tris-HCL, pH 7.4, 10 mM NaCl, 3 mM MgCl2, 0.1% IGEPAL CA-630) and immediately centrifuged at 550 X g for 10 minutes at 4 °C. The supernatant was discarded, cells were placed on wet ice, and the TDE1 Tagment DNA Enzyme and Buffer Kit (Illumina, #20034197) was utilized according to the manufacturer's instructions. Briefly, chondrocytes were incubated in the TDE1 reaction buffer for 45 minutes at 37°C, followed by addition of 10 µl of 3 M sodium acetate to stop the reaction. Nucleic acid purification was performed using the Qiagen MinElute Kit (#28204) following the manufacturer's instructions. The transposed DNA was PCR amplified and indexed using the Illumina Nextera Kit (Illumina) and NEBNext High-Fidelity 2x PCR Master Mix (NEB) for 12 cycles to generate each library. The PCR reaction was subsequently purified using AMPureXP beads (Agencourt).