title: GSM9278854: TET2KO U937 cells expressing doxycycline-inducible TurboID OGT, 18hr induction dox 1ng/ml, IP: Strepavidin; Homo sapiens; ChIP-Seq
library_construction_protocol: Briefly, cells were double cross-linked with disuccinimidyl glutarate (Thermo) and formaldehyde for 30 minutes at room temperature and quenched with 2.5M glycine. Cross-linked cells were treated with LB1 buffer (50mM HEPES-KOH, pH7.5, 140mM NaCl, 1mM EDTA, 10% Glycerol, 0.5% NP-40, 0.25% Triton X-100 in distilled water) x 10 minutes at 4C, LB2 buffer (10mM Tris-HCl, pH 8.0, 200mM NaCl, 1mM EDTA, 0.5mM EGTA in distilled water) x 10 minutes at 4C, then sonicated in LB3 buffer (10mM Tris-HCL, pH 8.0, 100mM NaCl, 1mM EDTA, 0.5mM EGTA, 0.1% Na-deoxycholate, 0.5% Na-Lauroylsarcosine, 1% Triton X-100, in distilled water) using Covaris, E220. Following antibody incubation overnight, immunoprecipitation (IP) was performed using Protein-A or Protein-G Dynabeads (Thermo) or Strepavidin magnetic beads (Thermo 88817) for 1 hour. Beads were collected via a magnet and washed six times with RIPA buffer (50mM Hepes-KOH, pH 7.5, 500mM LiCl, 1mM EDTA, 0.7% Na-deoxycholate, 1% NP-40 in distilled water), twice with buffer 500 (0.5g deoxycholic acid, 1mM EDTA, 5mM Tris-HCl, pH 8.1, 1% Triton X-100, and 0.02% NaN3 in distilled water), and twice with LiCl buffer (2.5 g deoxycholic acid, 1 mM EDTA, 250 mM LiCl, 0.5% NP-40, 10 mM Tris-HCl pH 8.1, 0.02% NaN3 in distilled water). Beads were washed briefly with TE buffer and DNA was eluted in ChIP elution buffer (50mM Tris-HCl, pH 8.0, 10mM EDTA, 1% SDS in distilled water) and de-crosslinked at 65C for 2 hours. DNA was purified by column purification (DNA Clean and Concentration, Zymo Research). Library preparation was done according to manufacturer's instructions (ThruPlex DNA-Seq Kit, Takara or NEBNext® Ultra, NEB).
identifiers.namespace: GEO
identifiers.id: GSM9278854_r1