title: GSM4801092: DKO0_Uhrf1_Input_rep1; Mus musculus; ChIP-Seq
library_construction_protocol: 5 (histone) or 25 (Trim28) million cells were crosslinked with 1% FA for 5 or 8 minutes respectively at RT. Glycine was added to a final concentration of 125mM and mixed for 5 minutes at RT. Crosslinked cells were washed with DPBS 2x then spun down 3min 15000g.  Cells were then incubated with 500µL cell lysis buffer (20 mM Tris-HCl ph8.0, 85 mM KCl, 0.5% NP 40) for 10 minutes on ice then spun down for 3min at 2500g. Supernatant was removed and the cell pellet was resuspended in 500µL of nuclear lysis buffer (10 mM Tris–HCl, pH 7.5, 1% NP-40, 0.5% sodium deoxycholate, 0.1% SDS) then incubated for 10 minutes on ice. Volume was increased to 1mL using Nuclei Lysis Buffer then sonicated on a Covaris E220 Evolution sonicator (PIP = 140.0, Duty Factor = 5.0, Cycles/Burst = 200, 10minutes). After sonication, chromatin was spun down at 15000g for 10 minutes to pellet insoluble material. Volume was increased to 1.5mL with Chip Dilution Buffer (0.01% SDS, 1.1% Triton X-100,1.2mM EDTA, 16.7mM Tris-HCl pH 8.1, 167mM NaCl) and 2ug of H3K4me3 antibody (Abcam: ab8580), H3K36me3 (Active Motif 61101), H3K9me3 (Abcam, ab8898), or Trim28(Abcam, ab22553) were added. Immunoprecipitation mixture was allowed to rotate overnight at 4°C. The next day, 40µLs of Protein A Dynabeads (Thermo, 10001D) were added to the IP mixture and allowed to rotate for 4 hours at 4°C. This was followed by two washes of each: low salt wash buffer (0.1% SDS, 1% Triton X-100, 2mM EDTA, 20mM Tris-HCl  pH 8.1, 150mM NaCl); high salt wash buffer (0.1% SDS, 1% Triton X-100, 2 mM EDTA, 20mM Tris, pH 8.1, 500 mM NaCl); LiCl wash buffer (0.25M LCl, 1% NP40, 1% deoxycholate, 1mM EDTA, 10mM Tris-HCl pH 8.1); and TE buffer pH 8.0 (10mM Tris-HCl, pH 8.0, 1mM EDTA pH 8.0). DNA was eluted twice using 50µLs of elution buffer (0.5 to 1% SDS and 0.1 M NaHCO3) at 65°C for 15 minutes. 16µL of reverse crosslinking salt mixture (250 mM Tris-HCl, pH 6.5, 62.5 mM EDTA pH 8.0, 1.25 M NaCl, 5mg/ml Proteinase K) was added and samples were allowed to incubate at 65°C overnight. For library preparation, DNA was purified using AMPure XP beads (Beckman-Coulter) and treated with DNase-free RNase (Roche) for 30 minutes at 37 °C. DNA libraries were then end repaired and A-tailed using Ultra II End Repair/dA-Tailing Module (NEB) and adapters (Broad Institute, single index P7) were ligated using Blunt/TA Ligase Master Mix (NEB). Next, libraries were PCR amplified using Pfu Ultra II Fusion High-fidelity DNA Polymerase (Agilent) then size selected on a gel for fragments between 200-1000bp
attributes.tag: GEO Accession
attributes.value: GSM4801092