GSM4273029: WT_input; Mus musculus; ChIP-Seq
Cells were washed in cold PBS, fixed in 1% formaldehyde at room temperature for 10 min and the fixation reaction was quenched with 0.125M glycine for 5 min at room temperature. The nuclei were resuspended and lysed in 500µl of shearing buffer containing 0.5% SDS, 10mM EDTA pH8, 50mM Tris-HCl pH8, 5mM sodium butyrate and protease inhibitor. The chromatin was sheared on the Covaris E220 sonifiyer, according to the following program: PIP70, 5% duty factor, 200 cycles per burst, 25min. The sheared chromatin was diluted in 1% Triton, 2Mm EDTA, 150 Mm NaCL, 20Mm Tris-HCl pH8 plus protease inhibitor. A small portion of the shearded chromatin was set aside as total input, reverse crosslinked with 1 µl of 10mg/ml RNAase for 30 min at 37°C and 5µl of 20mg/ml Proteinase K at 65°C overnight, purified using the Qiagen PCR purification Kit and DNA is quantify on Qubit 3.0 fluorimeter. The immunoprecipitated chromatin was cleaned up using the Qiagen PCR purification Kit. Input and ChIP Libraries were prepared with the Rubicon ThruPLEX FD kit (ref. R400429, Takara Bio USA) and sequenced on Illumina NextSeq instrument to generate 75bp single-end sequencing reads.
GEO Accession
GSM4273029