IL22 induces genome-wide Stat3 binding in primary mouse hepatocytes in vitro [ChIP-Seq] (Mus musculus)
Primary mouse hepatocytes were treated with IL22 for 2 h. Subsequently, cells were cross-linked with 1% formaldehyde, and the reaction was quenched with glycine for 5 minutes. After washing in PBS, nuclear extracts were further generated using Sonication ChIP Kit (Cell Signaling Technology, 56383). Chromatin was fragmented using Cup-type Ultrasonicator (Scientz08-IIIC) (10 s on and 10 s off for 45 cycles). Chromatin fragments were incubated with p-Stat3 antibody (Cell Signaling Technology, 9145; 1:100 dilution), rabbit IgG (Cell Signaling Technology, 2729; 1:100 dilution) was used as a control. Antibody-chromatin complexes were captured using Protein A/G Magnetic Beads (MedChemExpress, Shanghai, China, #HY-K0202). The cross-links were then reversed, and the samples were sequentially treated with RNase and proteinase K. Finally, both input and ChIP DNA were purified using the AFTSpin Multifunction DNA Purification Kit (Diagenode, C03040001S), quantified with a Qubit fluorometer (Life Technologies), and used for library preparation. For ChIP-seq, libraries were constructed with the TruSeq Nano DNA High Throughput Library Prep Kit from Illumina (20015965) and sequenced on an Illumina sequencing platform in PE150 mode to generate 150-bp paired-end reads.