GSM9340420: Neutral Rev dMpp8 Clone13A; Mus musculus; OTHER
Cells were fixed with 3 mM DSG for 35 minutes, followed by an additional 10 minutes with 1% formaldehyde. Following quenching, nuclei were prepared from fixed cells. Chromatin was digested in situ using MNase, DNA 3' ends were chewed back and biotinylated, and then free DNA ends were proximity ligated. Unligated ends were digested, and followed by reverse crosslinking of the chromatin and Protease K digestion. DNA was purified by phenol chloroform extraction and DNA Clean and Concentrator kit (Zymo), and then size selected for dinucleosomal fragments on an agarose gel. Following end polishing of DNA fragments, a streptavidin pulldown was used to enrich for ligated dinucleosomal fragments. From bead-bound DNA fragments, Micro-C libraries were generated using NEBNext Ultra II DNA Library Prep (NEB, E7645S) on-bead following the manufacturer's instructions, and amplified by 8 cycles of PCR followed by 0.95x AMPure XP bead purification (Beckman Coulter).