GSM8864701: RNApolyII_L1; Homo sapiens; ChIP-Seq
HT1080 cells were seeded into 20 cm dishes. Formaldehyde was added directly to the medium to a final concentration of 1% and incubated for 20 minutes at room temperature. To quench the crosslinking reaction, 10x glycine was added for 5 minutes. The tubes were then sonicated on ice using a tip sonicator for 5 minutes (50% amplitude, 1-second on/1-second off intervals). The chromatin lysate was diluted with ChIP lysis buffer, and a 1% input sample was removed. Then, 5 μg of FOXO3a antibody (Affinity Biosciences, #AF3771) was added to the protein sample and incubated overnight at 4°C. Protein A/G magnetic beads were added and incubated for 2 hours at 4°C. The beads were sequentially washed with low salt, high salt, LiCl, and TE buffers. ChIP DNA was eluted at 62°C for 2 hours in 100 μl of elution buffer containing proteinase K, and the final products were obtained after purification (absin, #abs50034). ChIP product was used for library preparation. The ChIP product was treated with End Prep Enzyme Mix for end repairing, 5' Phosphorylation and dA-tailing in one reaction, followed by ligation to adaptors with a “T” base overhang. Adaptor_x0002_ligated DNA was then recovered using Beads. Each sample was then amplified by PCR using P5 and P7 primers, with both primers carrying sequences which can anneal with flowcell to perform bridge PCR and P7 primer carrying index allowing for multiplexing. The PCR products were cleaned up using beads, validated using an Agilent 2100 Bioanalyzer, and quantified by Qubit 3.0 Fluorometer.
GEO
GSM8864701_r1