GSM9201376: WT_NPC_CTCF_Rep1; Mus musculus; ChIP-Seq
Briefly, ~4 × 107 and ~1 × 107 cells were used for co-activators and histone marks, respectivel. Cells were crosslinked with 1% formaldehyde for 10 min at room temperature (RT) and quenched with 0.125 M glycine for 10 min. Cells were consecutively incubated with three different lysis buffers (Buffer 1_ 50 mM HEPES, 140 mM NaCl, 1 mM EDTA, 10% glycerol, 0.5% NP-40, 0.25% TX-100; Buffer 2_ 10 mM Tris, 200 mM NaCl, 1 mM EDTA, 0.5 mM EGTA; Buffer 3_ 10 mM Tris, 100 mM NaCl, 1 mM EDTA, 0.5 mM EGTA, 0.1% Na-deoxycholate, 0.5% N-lauroylsarcosine) in order to isolate chromatin. Next, chromatin was sonicated for 8 minutes in Buffer 3 (20 s on, 30 s off, 25% amplitude) using an EpiShear probe sonicator (Active Motif).  The sonicated chromatin was mixed with 3 μg of H3K27ac antibody or 10 μg of Rad21 antibody and incubated overnight at 4°C. Next, 50-100 μl of Dynabeads (Thermo Fisher, 10-002-D) were aliquoted into a microtube for each ChIP reaction. The dynabeads were washed with 1 ml of cold blocking solution (0,5% BSA and 1x PBS). The antibody bound chromatin was added to the washed beads and incubated for four hours at 4ºC. Magnetic beads were washed five times with RIPA buffer (50 mM Hepes, 500 mM LiCl, 1 mM EDTA, 1% NP-40 and 0,7% Na-Deoxycholate). The chromatin was eluted and incubated at 65 °C overnight to reverse the crosslinking. Next, samples were treated with 0,2 mg/ml Rnase A and 0,2 mg/ml proteinase K. DNA  purification was performed using Zymoclean Gel DNA Recovery kit (Zymo Research, D4008). TruSeq DNA Sample prep Kit (Illumina) ; starting material 10ng