GSM5741228: ChIP-seq_Foxa1_W2; Mus musculus; ChIP-Seq
Three mouse prostates were dissected and minced with scissors in 5 ml cold DPBS with PMSF. Shredded tissue was fixed with 1% formaldehyde at room temperature for 10 min with rotation. Then, 125 mM glycine was added to quench the formaldehyde at room temperature for 5 min. 3ml of ice-cold lysis buffer (1% SDS, 10 mM EDTA, 50 mM Tris-HCl, 1× proteinase inhibitor) was added and incubated at 4 °C for 30 min 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: 15 min). Clarified samples were collected by centrifugation at 15,000 rcf for 15 min at 4 °C. After preclearing with 30 μL of protein G beads (Invitrogen, 10003D), 3 μL of anti-FOXA1 (Abcam ab23738)/FOXA2 (Abcam ab256493) antibody was added for immunoprecipitation overnight. To bind the anti-FOXA1/FOXA2 antibody, 100 μL of protein G beads was added and incubated with rotation for three 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 of elution buffer. The samples were then digested with 10 μL of Proteinase K (Invitrogen, 25530049) with incubation at 65 °C for 5 h. DNA was purified with DNA Clean & ConcentratorTM-5 (Zymo Research, D4004). Two nanograms of eluted DNA was used as input for library construction with a TruePrep DNA Library Prep Kit V2 for Illumina (Vazyme, TD503). The libraries were sequenced with the Illumina NovaSeq sequencing system (PE 2×150 bp reads) at Berry Genomics.