Truchlp tissue chromatin shearing kit

Chromatin was prepared from up to 50 mg jejunum using SDS Shearing Buffer and the truChlP Tissue Chromatin Shearing Kit (Covaris, Brighton, UK). Chromatin immunoprecipitations (ChIPs) were run on the IP-Star compact system using the Auto iDeal ChIP-seq kit for histones (Diagenode, Seraing, Belgium). For ChIP-seq validation, shredded intestinal tissue was cross-linked in 1% formaldehyde for 8 min at room temperature and homogenized in ZR 2.0 mm BashingBead™ Lysis Tubes (Zymo Research Corp., Tustin, USA) with a Precellys24 homogenization device (VWR International GmbH, Darmstadt, Germany). ChIP assays were performed with minor modifications as described [15 (link)]. DNA from 15 mg tissue was used in each IP with H3K4me3, H3K27me3, H3K36me3, and H3pan premium rabbit polyclonal antibodies (Diagenode). A non-targeting IgG rabbit antibody (Cell Signaling, Leiden, Netherlands) was used to control non-specific signals. One microliter of purified chromatin was applied for semi-quantitative real-time PCR using the absolute qPCR SYBR Green Mix (Thermo) performed with the ABI 7500 Real-Time PCR System (Applied Biosystems, Forster City, USA). Primers are given in the Additional file 1. All PCR results were normalized to input controls and to an unmodified region (background).

Chromatin was prepared up to 50-mg jejunum using SDS shearing buffer and the truChlP Tissue Chromatin Shearing Kit (Covaris, Brighton, UK) according to the manufacturer’s instructions. ChIPs were run on the IP-Star compact system using the Auto iDeal ChIP-seq kit for histones (Diagenode, Seraing, Belgium) according to the manufacturer’s direct method for ChIP preparation.