GSM7750299: CAMA-1 cf-chromatin, cfMNase-Seq, 5 min MNase digestion; Homo sapiens; OTHER
Conditioned media was collected in exponential growth phase. The media was spun at 2500g for 10 min at 4 °C, transferred to a new tube, and spun again at 16100g for 10 min at 4 °C. The supernatant was transferred to a new tube and kept at 4 °C until MNase digestion. Processed media samples were subject to either no MNase digestion, or a 1, 2, 5, 10 or 30 minute digestion, using an MNase master mix. The MNase master mix was prepared at 3000 Units/mL (U/mL) by combining 440 μL of ddH20, 50 μL of MNase buffer, 5 μL of BSA, and 5 μL of 300,000 U/mL MNase (New England Biolabs, CAT #M0247S). MNase was added to the processed media samples at a final concentration of 20 U/mL and incubated in a 37 ºC water bath for corresponding digestion time. The reaction was halted with 5 mM EDTA (Invitrogen, CAT #15575020). After Mnase digestion, purification of DNA from cell-free chromatin was performed using the Qiagen Circulating Nucleic Acid kit (CAT #55114). The DNA was quantified using the Qubit High Sensitivity dsDNA kit (Life Technologies, CAT #Q33231) and analyzed with a Agilent 2100 Bioanalyzer using the High Sensitivity DNA kit (CAT #5067-4626). Libraries were prepared from 20 ng of purified DNA using the KAPA Hyperprep Kit with Library Amplification Primer Mix (Roche, CAT #07962363001) and adapters containing unique molecular identifiers from Integrated DNA Technologies. The libraries were cleaned-up using Beckman Coulter AMPure XP beads (CAT #A63882) with a 0.8× cleanup before library amplification and a 0.9X cleanup afterward. The libraries were amplified with six PCR cycles. Quality control was performed with a Qubit High Sensitivity dsDNA kit and  Agilent 2100 Bioanalyzer.