GSM4432402: MTCH2_KD-Input; Homo sapiens; ChIP-Seq
Lysates were clarified from sonicated nuclei and histone-DNA complexes were isolated with H3K27ac antibody. Samples were prepared as outlined by Takara Bio USA Inc's ThruPLEX DNA-seq Kit user guide. Briefly, samples were normalized to 10ng of DNA input and topped up to 10ul with nuclease-free water. Samples were then mixed with 3ul of template preparation master mix and incubated at 22°C for 25 minutes, then 55°C for 20 minutes in a Veriti 96-well thermocycler (Thermo Fisher). Following template preparation, samples were mixed with 2ul of library synthesis master mix and incubated at 22°C for 40 minutes on the same cycler. Samples were prepared for library amplification as outlined in the user guide and indexed individually. From here, PCR cycles were optimized by adding 0.75ul of 10x SYBR green I nucleic acid gel stain (Thermo Fisher) to each sample. Once mixed, 10ul from each sample was removed and run on a CFX96 Touch Real-Time PCR cycler (Bio-Rad). Samples were normalized to 1/3 of their amplification curve and amplified on a Veriti 96-well thermocycler. Samples were topped up to 50ul with nuclease-free water prior to bead cleanup with AMPure XP beads (Beckman Coulter). Final library sizing and QC was evaluated on Agilent's high sensitivity DNA kit run on the 2100 Bioanalyzer (Agilent Technologies).