GSM2070528: MORC3_ChIPseq_input_replicate 2; Mus musculus; ChIP-Seq
Cells were fixed by treatment with 1% Formaldehyde and 1xPBS for ten minutes at room temperature, then quenched by addition of glycine to a final concentration of 0.14 M and incubated a further ten minutes. Cells were washed with 1xPBS, then aliquoted and flash frozen.  Ten million cells per replicate were thawed on ice and resuspended in 1 mL 10 mM Tris pH 8.0, 0.25% Triton X-100, 10 mM EDTA, 0.5 mM EGTA, and 1 mM PMSF and incubated with rotation for fifteen minutes at room temperature. Nuclei were pelleted by centrifugation at 1500g for five minutes at 4 °C. The nuclei were resuspended in 10 mM Tris pH 8.0, 200 mM NaCl, 10 mM EDTA, 0.5 mM EGTA, and 1 mM PMSF, and incubated for ten minutes at room temperature with rotation, and again centrifuged. Nuclei were then resuspended in 10 mM Tris pH 8.0, 10 mM EDTA, 0.5 mM EGTA, 0.1% SDS, and 1 mM PMSF, and disrupted by sonication at high intensity using a Bioruptor (Diagenode).  Sonicated lysate was cleared by centrifugation at 16,000xg for ten minutes, and the supernatant was used for ChIP. Samples were diluted with an equal volume 16.7 mM Tris pH 8.0, 0.01% SDS, 1.1 % Triton, 1.2 mM EDTA, and 167 mM NaCl. The samples were then pre- cleared with 30 μL Protein A magnetic Dynabeads (Thermo Fisher Scientific) (which had been washed with 16.7 mM Tris pH 8.0, 0.01 % SDS, 1.1 % Triton X-100, 1.2 mM EDTA, and 167 mM NaCl prior to use) and incubated for two hours at 4 °C. The beads were then collected on a magnet and the supernatant retained. Ten percent of the sample was saved for input. The sample was then split into two halves; one half was treated with 1 μL rabbit IgG while the other half was incubated with 1 μL anti-MORC3 antibody (100-401-N96S, Rockland). Samples were incubated overnight at 4 °C with rotation.  60 μL of Protein A beads (which had been washed with 16.7 mM Tris pH 8.0, 0.01 % SDS, 1.1 % Triton, 1.2 mM EDTA, and 167 mM NaCl prior to use) were added to each sample and incubated for a further two hours. The beads were washed twice, four minutes each with rotation with 50 mM HEPES pH 7.9, 1% Triton X-100, 0.1% deoxycholate, 1 mM EDTA, and 140 mM NaCl, and then washed twice, four minutes each with rotation with 50 mM HEPES pH 7.9, 0.1 % SDS, 1 % Triton X-199, 0.1% deoxycholate, 1 mM EDTA, and 500 mM NaCl, and then washed twice, four minutes each with rotation with 500 μL 10 mM Tris pH 8.0 and 1 mM EDTA. The purified DNA was eluted by incubation with elution buffer (100 μL 50 mM Tris pH 8.0, 1 mM EDTA, and 1% SDS) at 65 °C for ten minutes. Eluent was collected on a magnetic rack, and then the beads were resuspended with 150 μL elution buffer and incubated at 65 °C for ten minutes. The two eluents were pooled and de-crosslinked by incubation at 65 °C overnight.  The samples were brought to room temperature and then warmed to 37 °C and then incubated with 10 μg RNase A. Samples were then treated with 15 μg Proteinase K and incubated for two hours at 56 °C. Finally, the samples were purified with Qiagen MinElute columns. Purified DNA was quantified with Qubit High Sensitivity reagent (Thermo Fisher Scientific) and libraries were generated with the Ovation Ultralow Library System kit (Nugen) using 10 ng input DNA.