ChIP-Seq p65 of mus musculus: ILC2_IL33_rep1
At least 5 million cells were used for chromatin immunoprecipitation. After stimulation, cells were fixed with 1% formaldehyde (Sigma) for 10 min at room temperature followed by quenching with 0.125 M glycine (Sigma) for 10 min and PBS washing. Fixed cells were lysed in lysis buffer (10mM Tris pH8, 1mM EDTA pH8, 140mM NaCl, 1% Triton-X, 0.1% SDS, 0.1% Na-DOC) containing freshly added 1mM DTT and protease inhibitors (aprotinin, leupeptin, pepstatin, and PMSF) by incubating on ice for 10 min. Lysates were sonicated using the Q800R3 Sonicator (QSonica) and centrifuged to remove debris and to clear the lysate before immunoprecipitation with anti-p65 (Diagenode) overnight at 4 C. IP samples were then washed and incubated with RNAse for 30 min at 37 C followed by a proteinase K treatment for 2h at 37C. Beads were then decrosslinked overnight at 65 C and the DNA fragments were isolated using the AMPure XP beads (Beckman Coulter). Libraries were generated purified from purified DNA by using Ultra II DNA Library Prep Kit (NEBNExt) according to the manufacturers protocol. Libraries were sequenced by Novogene using Illumina NovaSeq 6000 for 150bp paired-end sequencing.