GSM9578924: shA3B 01 CIN 612 Input 1; Homo sapiens; ChIP-Seq
1 × 107 cells were harvested and collected in Southern lysis buffer before being treated with RNase A (5 ng/mL) and Proteinase K (7.5 ng/mL) at 37 °C overnight. DNA was purified from these samples using phenol-chloroform extractions, and 25 to 50 mg of DNA was used for each sample. DNA was sheared using a Bioruptor (Diagenode) on high power, 30 s on/90 s off cycles for 20 min. Input DNA was removed before loading the samples into preblocked magnetic beads in IP buffer containing 2 ug of the S9.6 antibody. Immunoprecipitations were allowed to incubate overnight at 4 °C while rotating. The next day, samples were washed 8 times with RIPA buffer for 5 min while rotating. One wash in TE buffer was performed before samples were eluted for 10 min at 65 °C in 10% sodium dodecyl sulfate (SDS), 10 mM Tris pH 7.4, 50 mM ethylenediaminetetraacetic acid (EDTA). DNA was purified from these elutions using a PCR purification kit (Qiagen) and stored at −20 °C. Samples were stored at -80° C until being shipped to Admera Biosciences (NJ), who performed the sequencing experiments. Briefly, the library was prepared using a KAPA HyperPrep Kit (Kapa Biosystems) following the manufacturer's recommendation. Input DNA was end-repaired and 3'-dA tailed. Adapter was then ligated to the DNA, and the ligated product was PCR amplified and cleaned up using the SPRIselect Reagent (Beckman Coulter). Quality control was then performed for the final library, followed by sequencing.