GSM6449871: 2273_CEBPA_ChIP; Homo sapiens; ChIP-Seq
Up to 2x10^7 cells (depending on the antibody used and the expression level of the TF of interest) were washed 3 times in PBS and resuspended in 10 mL of PBS. To achieve protein- protein cross-linking, cells were incubated with 0.25M Di(N-succinimidyl) glutarate (DSG) (Sigma-Aldrich) for 45 min on rotation at RT. Cross-linked cells were pelleted and washed 3 times with 10 mL of PBS, spinning at 300xg for 5 min at 4°C between washes.  For single crosslinking the pellet was resuspended in 10 mL of PBS. Proteins were fixed to chromatin by adding 16% formaldehyde (Pierce) to a final 1% concentration and incubating on rotation for 10 min at RT. To quench the cross-linking reaction, cells were incubated on ice for 5 min with 0.4M glycine (Sigma-Aldrich). Cross-linked cells were pelleted and washed twice with 50 mL of ice-cold PBS, spinning at 300xg for 5 min at 4°C between washes.  Cell pellets were resuspended in 10 mL of ice-cold lysis buffer A (10 mM HEPES pH 8.0, 10 mM EDTA pH 8.0, 0.5 mM MEGTA pH 8.0, 0.25% Triton-X 100) with the addition of 1x Protein Inhibitor Cocktail (PIC) (Sigma-Aldrich) and incubated on rotation for 10 min at 4°C. Residual nuclei were pelleted at 500xg for 10 min at 4°C, resuspended in 10 mL of ice-cold Buffer B (10 mM HEPES pH 8.0, 200 mM NaCl, 1 mM EDTA pH 8.0, 0.5 mM MEGTA pH 8.0, 0.01% Triton-X 100) with the addition of 0.1x PIC and incubated on rotation for 10 min at 4°C. Chromatin was pelleted at 500xg for 5 min at 4°C and resuspended to a final concentration of 5x10^6 cells/300 μL of ice-cold IP Buffer I (25 mM Tris-HCl, pH 8.0, 150 mM NaCl, 2 mM EDTA, pH 8.0, 1% Triton-X 100, 0.25% SDS) with the addition of 0.1x PIC.  A maximum of 300 μL of chromatin suspension were aliquoted in Bioruptor tubes (Diagenode) and fragmented by using a Bioruptor Pico Sonicator (Diagenode). For each cell type, the number of sonication cycles was optimized to obtain chromatin fragment ranging between 200 and 1000 bp. Each cycle was 30 sec long with a 30 sec break. Sonicated chromatin was spun down at 16000xg for 15 min at 4°C and resuspended in 2 volumes of IP Buffer II (25 mM Tris-HCl, pH 8.0, 150 mM NaCl, 2 mM EDTA, pH 8.0, 1% Triton-X 100, 7.5% Glycerol) with the addition of 1x PIC. Separated reactions were pooled and 10% of the chromatin suspension was stored as input control.  The following IP protocol is optimized for a maximum input of 5x10^6 cells, and multiple reactions were set up for experiments requiring a greater number of cells. For each IP, 10 μL of Dynabeads Protein G (Thermo Fisher) were washed once with 200 μL of 0.1M citrate phosphate pH 5.0 and twice with 200 μL of 100 mM NaH2 pH 8.0 phosphate buffer. 4 μg of C/EBPα antibody (sc-1662587), RUNX1 antibody (Ab23980) or c-Fos antibody (MA5-15055, Invitrogen) were added to 5 μL of 100 mM NaH2 pH 8.0 phosphate buffer, 0.5% BSA and incubated with Dynabeads Protein G on rotation for 2 hours at 4°C.  Chromatin was incubated with the antibody-beads mixture for 4 h (C/EBPα and RUNX-1 IPs) or overnight (c-FOS IPs) on rotation at 4°C. Beads were washed once with Wash Buffer I (20 mM Tris-HCl, pH 8.0, 150 mM NaCl, 2mM EDTA pH 8.0, 1% Triton-X 100, 0.1% SDS), twice with Wash Buffer II (20 mM Tris-HCl, pH 8.0, 500 mM NaCl, 2mM EDTA pH 8.0, 1% Triton-X 100, 0.1% SDS), once with Lithium Chloride (LiCl) Buffer (10 mM Tris-HCl, pH 8.0, 250 mM LiCl, 1mM EDTA pH 8.0, 0.5% NP-40, 0.5% Na-Deoxycholate) and twice with TE/NaCl Buffer (10 mM Tris-HCl, pH 8.0, 50 mM NaCl, 1 mM EDTA pH 8.0). Chromatin was eluted from beads in 100 μL of freshly prepared Elution Buffer (EB) (100 mM NaHCO3, 1% SDS) incubating on a shaker for 30 minutes at RT. Samples, including the input control, were incubated overnight at 65°C in 500 mM NaCl and 500 μg/ml proteinase K (Roche) to reverse crosslinks and digest proteins. After this step, multiple reactions were pooled together.  For quality control, samples were incubated with 1.8x volumes of AMPure XP beads (Beckman Coulter) for 30 minutes at RT. After washing beads twice with 200 μL of ethanol, chromatin was eluted in 50 μL of 0.1x TE pH 8.0.  The quality of the ChIP was assessed by qPCR. The immunoprecipitated DNA and the input control were amplified using primers specific for both putative binding sites of the TF in exam as well as non-bounding sites (Table 2.8) and the enrichment of the target sequences was measured over the input. ChIP library preparation was performed by using the KAPA Hyper Prep Kit (Roche) with a 200-450 bp library size selection.