GSM3692159: 2018001-atf4 (ChIP-seq); Homo sapiens; ChIP-Seq
Chromatin was sonicated by Bioruptor (Diagenode, Belgium) under the condition as follows: 20 cycles of 30 s on, 60 s off. The sonicated chromatin was spun down at 12,000 rpm for 10 min at 4 °C to collect the chromatin. Add antibody (10 μg) to the soluble chromatin (80 μg), and incubate at 4 °C with rotation overnight. Protein A/G Dynabeads (10 μl of beads per 1 μg of antibody, Life Tech, 10004D) were washed three times with Low Salt Wash Buffer. The washed Dynabeads were added to the soluble chromatin and antibodies, incubated at 4°C for 4 h with rotation. The magnetic Dynabeads were pelleted by placing the tubes in a magnetic rack and were sequentially washed once with Low Salt Wash Buffer, once with High-salt Wash Buffer, once with LiCl Wash Buffer. Wash the beads three times with TE buffer. Remove any supernatant remaining after the last washing. The beads were resuspended in 150 μl of Elution Buffer (50 mM Tris•Cl pH 8.0, 10 mM EDTA, 1% SDS), followed by incubation for 15 min at 65 °C. Repeat step 7 again, combine the elution, then you have 300 μl of the eluted DNA solution. Add 1 μl of high concentration RNase A (10 mg/ml) to the eluted DNA solution and the Input samples (500 μl), respectively. Incubate them at 37 ℃ for 1 h. Reverse formaldehyde crosslinks by respectively adding 12.5 μl or 55 μl of 5 M NaCl to the eluted DNA solution and the Input samples to a final concentration of 0.2 M. Incubate samples at 65 ℃ (650 rpm) for more than 8 h (less than 16 h).  Add both 80.5 μl of ddH2O and 5 μl of 20 mg/ml Proteinase K to the eluted DNA solution, and just 5 μl of Proteinase K to the Inputs. Incubate at 56 ℃ for 2 h. Extract once with 400 μl of phenol/chloroform/isoamyl alcohol, and once with 400 μl of chloroform/isoamyl alcohol (optional). Transfer 355 μl of supernatant to a new tube. Add 55 μl or 40 μl of 3 M NaAc (pH 5.2, 0.3 M final) to the Input samples and eluted DNA solution, 5 μl of glycogen each sample and mix well. Add 800 μl (two-fold volume) of absolute ethanol. Precipitate at -20 ℃ overnight or at -80 ℃ for 4 h. Centrifuge at 14000 rmp for 20 min at 4 ℃. Wash once with 75% ethanol and store at -20 ℃. The library is constructed under the protocol of Iillumina
GEO Accession
GSM3692159