GSM8464824: HCT116, TDG-KD, Valine deprivation, ddC-S1-END-seq; Homo sapiens; OTHER
4×106 HCT116 cells were plated in a 6 cm plate prior to the experiment. Cells were valine deprivation for 24 hrs and treated ddC (20 µM) for 20 hrs prior to harvest washed three times with cold PBS buffer Cells were collected and gently pipetted 5-10 times and transferred to a conical 15ml tube. Cells were neutralized with 10ml of cold PBS/EDTA 5mM/10% FBS buffer, then pelleted, resuspended in 1ml of neutralization buffer and transferred to an Eppendorf tube. They were then processed for END-seq or S1-END-seq. For S1-END-seq, cells were collected and embedded in 1% agarose plugs, lysed and digested with Proteinase K (1 hour at 50°C, followed by 7 hours at 37°C), washed with TE buffer, and then treated with RNAse A for 1 hour at 37°C. Plugs were then washed in EB and equilibrated in S1 nuclease buffer (40 mM sodium acetate pH 5.2, 300 mM NaCl, 2 mM ZnSO4) for 30 minutes. 1.8 U of S1 nuclease was added to 100 µL of S1 nuclease buffer per plug and incubated on ice for 15 minutes to allow for the enzyme to diffuse into the plug. The reaction mix was then placed at 37°C for 20 minutes before addition of EDTA (10 mM final concentration) to terminate the reaction Finally, plugs were processed through the standard END-seq protocol