GSM4983056: AR_ChIP-seq in LNCaP; Homo sapiens; ChIP-Seq
ChIP-seq was performed as previously described. In brief, 10 million cells were fixed with 1% formaldehyde at room temperature for 10 minutes with rotation. Then, 125 mM glycine was added to quench the formaldehyde at room temperature for 5 minutes. Cells were washed with cold PBS. The supernatants were then removed, and 880 μL of ice-cold cell lysis buffer (1% SDS, 10 mM EDTA, 50 mM Tris-HCl, 1× proteinase inhibitor) was added and incubated at 4°C for 30 minutes with rotation. An 880 μL cell lysate was transferred into a Covaris milliTUBE 1 mL AFA Fiber vial and sheared in a Covaris S220 ultrasonicator (fill level: 10, duty cycle: 5, PIP: 140, cycles/BURST: 200, time: 4 minutes). Clarified samples were collected by centrifugation at 16,100 rcf for 15 minutes at 4°C. After preclearing using 20 µL of protein G beads (Invitrogen, 10003D), 3 µL of anti-AR antibody was added (Abcam, ab108341) for immunoprecipitation overnight. To bind the anti-AR antibody, 60 μL of protein G beads was added and incubated with rotation for two hours at 4°C. The beads were washed twice each with Low Salt Wash Buffer, High Salt Wash Buffer and LiCl Wash Buffer and resuspended in 100 μL of freshly prepared DNA Elution Buffer (50 mM NaHCO3 and 1% SDS). The ChIP sample beads were placed on a magnet, and the supernatant was collected into a new tube. The above elution step was repeated with another 100 μL volume of elution buffer. The samples were then digested with 10 μL of Proteinase K (Invitrogen, 25530049) with incubation at 67°C for 4 hours. DNA was purified with DNA Clean & ConcentratorTM-5 (Zymo Research, D4004). One nanogram of eluted DNA was used as input for library construction with a TruePrep DNA Library Prep Kit V2 for Illumina (Vazyme, TD503). Libraries were sequenced with the Illumina NovaSeq sequencing system (PE 2×150 bp reads) at Berry Genomics.