GSM7750287: SU-DHL-6 cf-chromatin, 30ng input, ChIP-Seq, H3K27me3, rep2; Homo sapiens; OTHER
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). Cell-free ChIP-Seq was performed on the media cf-chromatin using specific antibodies against H3K4me3 (Abcam, ab1012) and H3K27me3 (Cell Signaling Technology, 9733S, lot 21), respectively. Libraries were prepared using an on-bead ligation protocol published in Sadeh et al (Nature Biotechnology, 2021). All library preparation steps were performed on Dynabead M270-Epoxy beads (Invitrogen, CAT #14301), up until the chromatin elution step was performed. Library preparation was otherwise conducted according to the KAPA Hyperprep Kit with Library Amplification Primer Mix (Roche, CAT #07962363001), utilizing sequencing adapters containing unique molecular identifiers (UMIs) 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. H3K4me3 libraries were amplified to 16 cycles, and H3K27me3 libraries were amplified to 12 cycles for 10 ng and 30 ng cf-chromatin input, and 11 cycles for 300 ng input. Quality control was performed with a Qubit High Sensitivity dsDNA kit and Agilent 2100 Bioanalyzer.