GSM5287384: Hoxb8_NFIL3_IP; Mus musculus; ChIP-Seq
Thirty million cells were crosslinked with 1% formaldehyde for 5 min, quenched with 1/20 volume of 2.5 M glycine and washed twice with PBS. To obtain nuclei, cell pellets were incubated in 1 mL Lysis buffer 1 (50 mM HEPES, pH 7.5, 140 mM NaCl, 1 mM EDTA, 10% glycerol, 0.5% NP40 and 0.25% Triton X-100) supplemented with protease inhibitors for 10 min, and 1 mL Lysis buffer 2 (10 mM Tris-HCl, pH 8.0, 200 mM NaCl, 1 mM EDTA and 0.5 mM EGTA) supplemented with protease inhibitors for 10 min. To shear chromatin, nuclei pellets were gently rinsed with Shearing buffer D3 (10 mM Tris-HCl, pH 7.6, 1 mM EDTA, and 0.1% SDS) twice, and then sonicated at 4 °C in Shearing buffer D3 for 17 min, of 10% duty cycle, 75 Watts intensity peak incident power and 200 cycles per burst, with an ME220 focused-ultrasonicator (Covaris). A 5% fraction of lysate were kept for input, and the remaining lysate were immunoprecipitated overnight at 4 °C with Dynabeads Protein G (Invitrogen) that had been pre-incubated with 5 mg of goat anti-NFIL3 antibody (sc-9550X; Santa Cruz Biotechnology). Beads containing protein-DNA complexes were washed and DNA fragments were reverse-crosslinked and eluted. Libraries were prepared with a ThruPLEX DNA-seq kit (Rubicon Genomics) and cleaned-up with AMPureXP.