title: GSM8506655: SK-N-BE(2)-C Palbociclib rep2; Homo sapiens; ChIP-Seq
library_construction_protocol: Cells were trypisinised and fixed in 1% formaldehyde for 10 minutes before quenching with 125mM glycine. Cells were rinsed with cold PBS plus protease inhibitor and lysed in 1ml of cold lysis buffer 1 (50mM HEPES-KOH; 140mM NaCl; 1mM EDTA; 10% glycerol; 0.5% Igepal CA-630; 0.25% triton X- 100) supplemented with cOmplete protease inhibitor then rotated at 4ºC for 10 minutes before being centrifuged at 2000g for 5 minutes at 4ºC. Pelleted nuclei were then resuspended in 1ml of cold lysis buffer 2 (10mM Tris-HCl; 200mM NaCl; 1mM EDTA; 0.5mM EGTA) supplemented with cOmplete protease inhibitor then rotated at 4ºC for 10 minutes before again centrifuging at 2000g for 5 minutes at 4ºC. Pelleted nuclei were then resuspended and lysed in 300μl of cold lysis buffer 3 (10mM Tris-HCl; 100mM NaCl; 1mM EDTA; 0.5mM EGTA; 0.1% Na-Deoxycholate; 0.5% N-lauroylsarcosine) supplemented with cOmplete protease inhibitor before being sonicated for 6 cycles in a Bioruptor 300 sonicator. 30μl of 10% Triton X-100 was then added to the lysate, and histone H2B and total mRNA added to final concentrations of 20ug/ml and 1ug/ml, respectively (acting as carrier). Lysate was centrifuged at 21000g for 10 minutes then supernatant was divided into 30μl for input, and 300μl for ASCL1 immunoprecipitation reaction. Replicate input samples for vehicle treated cells were pooled at an equimolar ratio and all sample libraries were prepared using the NEBNext Ultra II library prep kit. Libraries were then pooled and sequenced paired end to 150bp on a NovaSeq 6000