GSM6466266: CLL-Rat-Stat1; Rattus norvegicus; ChIP-Seq
Cells were fixed with 1% formaldehyde for 15 minutes. After quenching and cell lysis, chromatin was sonicated using a E220 focused-ultrasonicator (Covaris). Sonicated chromatin (~300 µg) was used for immunoprecipitation by incubation with Stat1 antibody (5 μg, Cell Signaling, #14994) overnight at 4 °C. ChIP Grade Protein A/G plus Agarose (20 μl, Thermo Fisher Scientific, #26156) were added to each ChIP reaction and reactions were incubated for 1 h at 4 °C. The beads were then incubated in 150 μl elution buffer at 65 °C for 40 min to elute immunoprecipitated materials. ChIP-seq libraries were prepared using the NEBNext ChIP-seq Library Prep Master Mix Set for Illumina (NEB catalogue number E6240L) and then run on the Illumina sequencer NovaSeq 6000. 10 ng of DNA was used as starting material for input and ip samples. Libraries were amplified using 13 cycles on the thermocycler. Post amplification libraries were size selected at 250-450bp in length using Agencourt AMPure XP beads from Beckman Coulter. Libraries were validated using the Agilent High Sensitivity DNA Kit.