GSM7986046: 293T WT-ZBTB9, ZBTB9, 0.1 Dox, rep2; Homo sapiens; ChIP-Seq
Cells were pelleted by centrifugation and resuspended in 8.25 ml of PBS and 8.25 ml of 2% formaldehyde and incubator for 10 minutes at room temperature. Cross-linking was quenched by the addition of 1665 µl of 1 M Tris/HCl pH 8.0 and 832.5 µl of 2.5 M glycine. Cells were pelleted and washed twice with PBS. Cross-linked cells were resuspended in LB1 Buffer (50 mM HEPES/KOH pH 7.5, 140 mM NaCl, 1 mM EDTA, 10% Glycerol, 0.5% NP-40, 0.25% Triton X-100 in distilled water) and rotated for 10 minutes at 4°C. Cells were pelleted and resuspended in LB2 (10 mM Tris/HCl pH 8.0, 200 mM NaCl, 1 mM EDTA, 0.5 mM EGTA in distilled water) and rotated for 10 minutes at 4°C. Cells were pelleted and resuspended in home-made RIPA Buffer (50mM Tris/HCl pH 8.0, 150 mM NaCl, 5 mM EDTA, 0.5% Na-deoxycholate, 0.1% SDS, 1% Triton X-100, in distilled water) sonicated (Covaris, E220). Samples were transferred into DNA LoBind tubes (Eppendorf) and clarified by centrifugation. Antibody-bead conjugates were made with either Protein-A (ThermoFisher). Beads were washed twice with home-made RIPA Buffer and resuspended with the appropriate amount of beads (according to manufacturer's instructions), and rotated overnight at 4°C. Antibody-bead complexes were applied to lysates and rotated overnight at 4°C. Beads were removed via magnet and washed twice with home-made RIPA Buffer , twice with Low salt wash Buffer (150 mM NaCl, 20 mM Tris-HCl, pH 8.0, 2 mM EDTA, 1% Triton X-100, and 0.1% SDS in distilled water), twice with High salt wash Buffer (250 mM NaCl, 20 mM Tris/HCl pH 8.0, 2 mM EDTA, 1% Triton X-100, and 0.1% SDS 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 8.0, 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 overnight at 65°C. DNA was purified by column purification (DNA Clean and Concentration, Zymo Research) and quantified by ScreenTape HS D1000 (Agilent) and Qubit (ThermoFisher). Library preparation was done using SMARTer® ThruPLEX® DNA-Seq Kit (Takara, #R400675).