hESC-VSMC ChIP-Input
ChIP-seq library preparation was performed as previously described. ChIP assays were performed in hESC-VSMCs or mouse aortic media with a SimpleChIP Enzymatic Chromatin IP Kit (#S9003, CST, USA) according to the manufacturer s instructions. The mouse aortic tissues were minced and grounded with a grinding rod for 1 min by adding liquid nitrogen and PBS. The aortic tissue or hESC-VSMCs were cross-linked with 1% formaldehyde (Sigma) at room temperature for 10 min. The reaction was quenched by an addition of 0.125 M glycine for 5 min, and the cross-linked chromatin was fragmented by 0.5l Micrococcal Nuclease (CST) for 20 min at 37  with sonication (Shanghai Lichen Bangxi Technology Co., Ltd.) for 3 times (20 s /time, peak power 250W, power ratio 20%). The sheared chromatin was then taken and 10% of which served as "Input DNA" and negative control. The rest was incubated with 10 l rabbit anti-human YY1 (Active Motif, Cat. No. 61779), rabbit anti-human H3K4me3 (abcam, ab8580), rabbit anti-human H3K27ac (abcam, ab4729), rabbit anti-human H3K27me3 (Millipore, #07-449), or mouse anti-human IgG antibody (1:1000 dilution, Millipore, #12-371), with rotation at 4C overnight. The reaction was subsequently pulled with ChIP-Grade protein G Magnetic Beads (CST, #9006). The protein G magnetic beads were then washed with low salt for 3 times (5 min/ time), and high salt for 1 time (5 min/ time). The ChIP elution buffer (CST, #7009) was added to the input sample and each ChIP sample for 30 min at 65  with gentle vortex, and then added 6 l 5M Nacl and 2 l Proteinase K (CST, #10012), and incubated 2 h at 65 C. 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).