Mab igg xp isotype control

CUT&RUN experiments were performed in two biological replicates using the Cell Signaling Technology CUT&RUN Kit following the manufacturer's instructions. Briefly, crypts from either EPR fl/fl or EPR cKO mice proximal colon were isolated and 5 mg of lightly fixed tissue (0.1% formaldehyde for 2 min at room temperature) were used for each experimental point. Upon incubation with Concanavalin A-coated beads and with freshly dissolved digitonin, cells were incubated (16 h at 4°C under rotation) with either anti-H3K27ac (Ab4729 from Abcam), anti-H3K27me3 (#9733 Cell Signaling Technology) or negative control rabbit (DA1E) monoclonal antibody (mAb) IgG XP^® Isotype Control (Cell Signaling Technology). pAG-MNase enzyme was added and activated to digest targeted regions of the genome. DNA was purified from input and enriched chromatin samples using the GFX PCR DNA and gel band Purification kit (Cytiva), and quantified prior to being utilized in qPCRs using the primers listed in Supplementary Table S2.

Cells were harvested by trypsinization, fixed with BD Cytofix/Cytoperm solution (BD Bioscience) and stained with Alexa Fluor^® 488-conjugated anti-Pan-CK antibody or corresponding mouse (DA1E) mAb IgG XP^® isotype control (1:50, Cell Signaling Technology) for 30 min at room temperature, followed by a flow cytometry analysis. The results were analyzed with Kaluza software (Beckman Coulter Inc.). The percentage of cells with Pan-CK positive represented the purity of cell lines^{15 (link)}. The experiments were independently performed for three times, and representative images were shown.

A chromatin immunoprecipitation (ChIP) assay was performed using a Magna ChIP G kit (# 17–409, MAGNA0002, Millipore), according to the manufacturer’s instructions. After crosslinking and sonication, 50 μL of sheared DNA was incubated with 20 μL of protein G magnetic beads and anti-β-catenin (1:50, #8480, Cell Signaling) at 4 °C overnight. Rabbit (DA1E) mAb IgG XP Isotype Control (1:50, #3900, Cell Signaling) was used as a negative control. Then, protein/DNA complexes were eluted and free DNA was purified for the following qRT-PCR assay. Promoter primers for the detection of gene downstream of Wnt signaling (CD44, CMYC, C-Jun) were designed, and the amplified products were confirmed to contain the β-catenin TCF binding site 5′-A/T A/T CAAAG-3′. The primer sequences are shown in Supplementary Table 1. qRT-PCR was performed as previously described [21 (link)].