ChIP-Seq of cells from islets of C57 mice without antibody (input)
The purified pancreatic islets or cell lines were cross-linked with 1% formaldehyde at room temperature for 10 minutes. The reaction was quenched by an addition of 0.125 M glycine for 5 minutes, and the cross-linked chromatin was fragmented by sonication (S220, Covaris) for 3 minutes (Peak power 175W, Duty 10%, Cycle per burst 200). The sheared chromatin was then diluted by 1:10 with dilution buffer, and 10% of which was taken to serve as "Input DNA" and negative control. The rest was incubated with 10 l of YY1 antibody according to the manufacturers protocol (Active Motif, Cat. No. 61779), and the chromatin/antibody complex was left to rotate end to end at 4C overnight. The reaction was subsequently pulled with protein G Dynabeads (Thermo Fisher Scientific). The immunoprecipitated genomic DNA was then washed, eluted and reverse cross-linked, followed by RNase and protease treatments. For ChIP-seq, the immunoprecipitated DNA was then resuspended in 20 l of water for library preparation. ChIP-seq libraries were prepared according to instructions of the NEBNext Ultra DNA Library Prep Kit for Illumina (NEB, Cat. No. E7645). Sequencing was performed at paired-end 149 bp on the Illumina HiSeq 2500 system according to manufacturers instructions. For ChIP-qPCR, the precipitated genomic DNA was resuspended in 50 l of water. qPCR was performed with 1 l of immunoprecipitated DNA or input DNA as template via iTaq Universal SYBR Green Supermix (BioRad, Cat No.1725122) on a CFX Connect Real-Time PCR Detection System (BioRad).