GSM2104363: HIRA Input; Mus musculus; ChIP-Seq
In parallel to sonication step, antibodies were coupled to magnetic beads (dynabeads InVitrogen, 112.03D, 112.01D) for at least 4 hours and washed 3 times in LB3. 10% of input was taken and protein–DNA complexes were selectively immunoprecipitated using WC15 antibody against HIRA, rolling overnight. Beads were then washed once with 20mM tris pH 8.0, 150mM NaCl, 0.1% SDS, 1% Triton, 2mM EDTA (WB1), once with 20mM Tris pH 8.0, 500mM NaCl, 0.1% SDS, 1% Triton, 2mM EDTA (WB2), once with 10mM Tris pH 8.0, 150mM LiCl, 1% NP-40, 1% DOC, 1mM EDTA, then TE 10:1, 50mM NaCl (WB3), and finally in TE 10:1. Samples were treated overnight with 50mM Tris pH 8.0, 10mM EDTA and 1% SDS at 65°C (elution buffer), to reverse cross-linking, then with RNAse (Qiagen, 19101) for an hour at 25°C and with PK for 2h at 56°C. DNA was purified with QIAGEN’s PCR purification kit (28104). Libraries were prepped using the NEB DNA Ultra kit, with bead bases size selection for an insert ~200bp