GSM9741527: EC ischemic limb, YY1 ChIP; Mus musculus; ChIP-Seq
Following skeletal muscle or lung tissue digestion, CD31+ ECs were isolated using CD31 MicroBeads (Miltenyi Biotec, 130-097-418) per the manufacturer's instructions. Briefly, cells were washed twice in cold, degassed buffer (PBS, 1% BSA, 2 mM EDTA) and centrifuged at 300g for 10 minutes to remove platelets. Up to 10⁷ cells were resuspended in 90 μL cold buffer, incubated with CD31 MicroBeads (10 μL per 10⁷ cells) for 15 minutes at 4°C, washed with 2 mL buffer, and resuspended in 500 μL buffer. Positive separation was performed on an autoMACS™ Pro Separator (Miltenyi Biotec); the magnetically retained fraction on MS Columns contained the CD31+ ECs. ChIP assays were performed using the SimpleChIP Enzymatic Chromatin IP Kit (Cell Signaling Technology, Cat. No. S9003) according to the manufacturer's instructions. Mouse muscle tissues were minced and enzymatically dissociated into single cells. The resulting muscle cells or hESC-ECs were cross-linked with 1% formaldehyde (Sigma) for 10 minutes at room temperature, and the reaction was quenched with 0.125 M glycine for 5 minutes. Crosslinked chromatin was fragmented with 0.5μL Micrococcal Nuclease (CST) for 20 minutes at 37 ℃, followed by sonication (Shanghai Lichen Bangxi Technology Co., Ltd.) for three cycles (20s each, peak power 250W, power ratio 20%). Ten percent of the sheared chromatin was saved as input DNA. The remainder was incubated overnight at 4°C with rotation with the respective antibody. Immune complexes were captured with ChIP-Grade Protein G Magnetic Beads (CST, Cat. No. 9006). Beads were washed three times with low-salt buffer (5 minutes each) and once with high-salt buffer for 5 minutes. ChIP Elution Buffer (CST, Cat. No. 7009) was added to input and ChIP samples and incubated at 65℃ for 30 minutes with gentle vortexing, followed by addition of 6 μL 5M NaCl and 2 μL Proteinase K (CST, Cat. No. 10012) and incubation at 65°C for 2 hours. For ChIP-seq, immunoprecipitated DNA was resuspended in 20 μL nuclease-free water and libraries were prepared with the NEBNext Ultra™ DNA Library Prep Kit for Illumina (NEB, Cat. No. E7645).