title: GSM7096112: WT, low MNase, Input, rep 2; Mus musculus; OTHER
library_construction_protocol: Cells were crosslinked using formaldehyde, lysed in hypotonic buffer and incubated with MNase for five minutes. The reaction was quenched using EDTA and samples were pre-cleared with protein A beads (NEB) in complete immunoprecipitation buffer (20mM Tris-HCl pH[7.5], 1M NaCl, 2M EDTA, 0.1% Triton X-100 with fresh protease inhibitors added). Input samples were taken from cleared chromatin. Pre-coupled antibody-protein A beads were added to cleared chromatin and incubated overnight at 4°C with rotation. Antibodies used were IgG (Abcam: ab37415, lot: GR3208186-1) and H3 (Abcam: ab1791, lot: GR3428015-1). Bead-bound samples were washed twice with low salt wash (20mM Tris HCl pH [8.0], 2mM EDTA, 150mM NaCl, 1% Triton X-100, 0.1% SDS) and then twice with high salt wash (20mM Tris HCl pH [8.0], 2mM EDTA, 500mM NaCl, 1% Triton X-100, 0.1% SDS). Samples were eluted at 65°C for 1.5 hours in elution buffer (100mM NaHCO3, 1% SDS) and cleaned using AMPureXP beads (Beckman Coulter). Cleaned DNA was used as a template for qPCR or for input into library builds. Library preparation was performed using NEB Next II Ultra DNA kit with 14 cycles of PCR amplification.