Chromatin Immunoprecipitation and RNA Sequencing

ChIP-seq were performed as described in previously [4 (link),6 (link)]. Briefly, cells were dual cross-linked with 2 mM ethylene glycol bis (succinimidyl succinate) (EGS) and disuccinimidyl glutarate (DSG) and 1% formaldehyde. Chromatin was isolated and then fragmented with 60 units MNase (New England Biolabs, M0247S) at 37 ⁰C for 10 minutes. For β-catenin ChIP-seq, cells were first fractionated by Subcellular Protein Fractionation Kit for Cultured Cells (ThermoFisher, Cat# 78840) to obtain a nuclear fraction that was diluted 1:5 with MNase digestion buffer (50 mM HEPES pH 7.9, 140 mM NaCl, 1% Triton X-100, 1 mM CaCl2, 1 mM DTT), and then digested with MNase as above. Antibodies used in the ChIP-seq experiments were against β-catenin (Cell Signaling, Cat#9581), WDR77 (SCBT, Cat# sc-100899), and MafB (SCBT, Cat# sc-10022).
For RNA-seq experiments, mRNeasy Mini Kit (Qiagen, No. 217004) was used for RNA extraction. 0.4 μg total RNA was used for RNA-seq library construction by a TruSeq RNA Library Prep Kit V2 (Illumina, RS-122-2001). Both ChIP-seq and RNA-seq libraries were sequenced at the Bauer Core Facility, Harvard.