Genome wide STAT3 binding analysis of primary human hepatocytes-derived organoids [ChIP-seq]

To analyze the primary STAT3 binding sites induced by OSM, chromatin immunoprecipitation sequencing (ChIP-seq) was performed using an anti-human STAT3 antibody. HHOs were cultured in EM for 1 week and cultured EM (-OSM) for 24 hrs to deplete OSM before OSM induction, and then incubated with EM(+OSM) for 0.5 hr. HHOs at 0h and 0.5h after OSM induction were subjected to ChIP-seq analysis using ChIPmentation methodology with minor adaptations [Schmidl et al. PMID: 26280331]. Briefly, organoids were dissociated into single cells and fixed for 10 min, and then quenched by glycine. The lysed and sonicated chromatin was transferred to a new tube. The chromatin was reacted with the anti-STAT3 antibody (Cell Signaling Technology: Stat3 (D3Z2G) Rabbit mAb #12640) on a rotator overnight at 4°C. Washed and blocked Dynabeads Protein A/Protein G magnetic beads in RIPA-LS supplemented with 0.1% BSA were added to the lysates and incubated for 2 hr on a rotator at 4°C. Beads were subsequently washed with RIPA-LS, RIPA-HS , RIPA-LiCl, and Tris-Cl pH 8.0. The beads were then resuspended in a tagmentation reaction mix containing 1 μl of TDE1 Tagment DNA Enzyme (Illumina) and incubated at 37°C for 3 min on a thermocycler. The beads were further washed and incubated with Pronase for 1 hr at 42°C and 8 hr at 65°C to revert cross-linking. Finally, DNA was purified with MinElute columns and eluted with elution buffer. The DNA was subsequently amplified with Nextera sequencing primers and NEBNext high fidelity 2x PCR master mix for 5–10 cycles. Enriched libraries were purified, size selected using AMPure XP beads to recover libraries with the fragment length of 200–400 bp, and sequenced by llumina HiSeq 2500 sequencer with 150 bp paired-end reads.