GSM5412046: 84577 [plus_young_H3K4me3]; Mus musculus; ChIP-Seq
Chromatin was prepared from 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. 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, US) with a Precellys24 homogenization device (VWR International GmbH, Darmstadt, Germany). ChIP assays were performed with minor modifications as described [Mannefeld et al. 2009]. DNA from 15 mg tissue was used in each IP with H3K4me3, H3K27me3, and H3pan premium rabbit polyclonal antibodies (Diagenode). A non-targeting IgG rabbit antibody (Cell Signaling, Leiden, Netherlands) was used as a control for non-specific signals. 1 µl of purified chromatin was applied for semi-quantitative real-time PCR using the absolute qPCR SYBR Green Mix (Thermo) performed with the ABI 7500 RealTime PCR System (Applied Biosystems, Forster City, US). Library preparations were performed according to the TrueSeq LT PCR free or TruSeq Nano DNA Kit instructions (Illumina, San Diego, USA). Sequencing was performed on the Illumina HiSeq 4000 with 76 bp paired-end sequencings.