title: GSM6923795: NC_Input; Homo sapiens; ChIP-Seq
library_construction_protocol: For crosslinking, the cells suspension was treated with formaldehyde at a final concentration of 1% (H3K56/vol) and incubated for 10 min at room temperature, followed by quenching with 0.125 M glycine for 5 min at room temperature. After pelleted through 1,000 g for 5 min at 4 °C, the harvested cells were washed twice with ice cold PBS, aliquoted to 20 million aliquots per 1.5 ml Eppendorf tube, and stored at -80 °C or directly for chromatin immunoprecipitation. 20 million crosslinked cells were lysed gently with 0.6 ml of ice-cold NP-40 lysis buffer (10 mM Tris·HCl, pH 7.5, 150 mM NaCl and 0.05% Nonidet P-40) on ice for 7 min. The cell lysates were then transferred on top of 1.25 ml sucrose cushion (24% sucrose (H3K56/vol) in NP-40 lysis buffer), centrifuged at 20,000 g, 10 min at 4 °C to discard the supernatant of cytoplasmic fraction. After washing once with 1ml PBS/1 mM EDTA, the chromatin pellet was resuspended in sonication buffer (20 mM Tris HCl, pH 8.0 ,150 mM NaCl, 2 mM EDTA, pH 8.0,0.1% SDS,1% Triton X-100) with 5 mM CaCl2. The suspension was treated with 400 U Micrococcal Nuclease and incubated at 37 °C for 15 min with 700 rpm shaking. The digestion was stopped by adding 20 μl 0.5 M EDTA and 40 μl 0.5 M EGTA and mixed thoroughly to inactivate MNase. The MNase digested chromatin were divided into 100 μl per tube and sonicated by the Bioruptor system (Diagenode, Bioruptor Plus) twice with high energy, at 30 s ON, 60 s OFF for 10 cycles. After centrifuged twice at 20,000 g, 10 min at 4 °C, 20 μl supernatant were sampled as input, and the rest supernatant was transferred into 2 new DNase free tube (600 μl/tube).   1 μg of myc-tag, H3K4me3, H3K27ac, GLYR1, BRD4, MED1 or Pol II antibody, was added for each ChIP reaction, respectively. After incubated overnight at 4 °C with low-speed rotation, the reaction mixture was treated with pre-washed 30 μl protein A/G magnetic beads by sonication buffer and incubated for another 4 h. The beads-antibody-protein complexes were washed through magnetic frame three times with sonication buffer, twice with high-salt wash buffer (20 mM Tris·HCl, pH 8.0, 500 mM NaCl, 2 mM EDTA, 0.1% SDS, 1% Triton X-100), twice with LiCl wash buffer (10 mM Tris HCl, pH 8.0,250 mM LiCl, 1 mM EDTA, 1% NP-40), three times with TE buffer (1 mM EDTA, 10 mM Tris HCl, pH 8.0). Washed bead-antibody-protein complexes were resuspended in 300 μl elution buffer (50 mM Tris HCl, pH 8.0,10 mM EDTA,1% SDS). In parallel, 250 μl elution buffer, and 5 μl 10% SDS were added to 50 μl of each input sample. In order to digest the proteins and reverse the crosslinking, 3 μl of Proteinase K solution (Invitrogen, 20 mg/ml) was added to the input samples and bead-antibody-protein complexes eluate, incubated overnight at 65 °C while shaking on the thermomixer, and the protease K was further inactivated at 80 °C for 20 min. The DNA was purified using Phenol:Chloroform:Isoamyl Alcohol 25:24:1 (pH8.0), precipitated using ethanol, and redissolved in DEPC water. Libraries from the purified ChIP-ed DNA were prepared using NEB Next Ultra II DNA library prep kit according to the manufacturer-supplied protocol, the 200~400 bp length amplified DNA were size selected using the SPRIselect double side size selection kit (Beckman Coulter), analyzed with Qubit assays and Fragment Analyzer, and finally sequenced on the HiSeq Xten PE150 sequencing platform (Novogene, Beijing).