GSM8426816: HDF1_shAR_LaminA/C ChIP; Homo sapiens; ChIP-Seq
Cells were fixed with 1% formaldehyde for 10 minutes. To determine chromatin binding of pSer301 and wild-type lamin A/C together with histone markers, chromatin was prepared using the Chromatin EasyShear Kit - Low SDS (Diagenode, Cat# C01020013) and sonicated using an E220 focused ultrasonicator (Covaris). The ChIP reaction was performed using the iDeal ChIP-seq kit (Diagenode, Cat# C01010055). Samples were pre-cleared using the beads provided in the kit and incubated overnight at 4°C with 5 µg of commercially available antibodies against lamin A/C (Abcam, #ab108595), phospho-Ser301 lamin A/C (Affinity, #AF7177), H3K27Ac (Abcam, #ab4729). Antibody-chromatin complexes were pulled down using protein A-coated magnetic beads. Elution was performed according to the manufacturer's instruction and chromatin was quantified using the Qubit Fluorometric Quantification Kit (Thermo Scientific). For library preparation, 10 ng of DNA was utilized with the NEBNext® ChIP-Seq Library Prep Reagent Set for Illumina. 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.