title: GSM5799953: in_Nutlin_pep_R2; Homo sapiens; ChIP-Seq
library_construction_protocol: The cells were fixed with 1% formaldehyde for 10 min and quenched with 125mM glycine for 5 min before nuclei isolation in NRO buffer. Shearing was performed based on the Covaris truChIP Chromatin Shearing Kit and sheared in 1 mL of Covaris D3 buffer for 8 min, with a duty factor 5% and peak power of 75,200 cycles/burst with a Covaris M220 Focused-ultrasonicator. Protein G Dynabeads (6 µg antibody 25 µL beads per condition.) were loaded with the crosslinked chromatin and allowed to incubate with shaking at 4°C overnight (~18hr). The beads were then washed at 4°C with 2x IP Buffer, 2x RIPA Buffer, 2x LiCl Wash Buffer and 2x TE Salt Buffer. Samples were kept on ice throughout the wash steps, and all washes were completed in less than 30 minutes; each wash step was 1 min to reduce sample loss.  After washes, the sample was eluted from beads with PK buffer containing 100 µg of Proteinase K and incubated at 65oC for 2 hr with periodic vortexing. The eluted sample was removed from the magnetic beads and reverse-crosslinked for 16 hr at 65°C. DNA was purified by phenol chloroform extraction using Light-5PRIME Phase Lock Tubes based on the manufacturer's instructions. To increase yield, samples were ethanol precipitated from glycogen (20µg/sample), sodium acetate (pH 5.2) and 10mM MgCl2 and kept at -20ºC overnight. Libraries with approximately 1 ng of input DNA were prepared using the KAPA Hyper Prep Kit and sequenced on Illumina NextSeq V2 high output 75-cycle. Adapters were diluted 1:10 before use and amplified for 12-14 cycles before sequencing. KAPA Hyper Prep Kit (KAPA Biosystems: KK8502)