GSM7070806: CUTTag_PD42_TEAD4; Homo sapiens; OTHER
The primary human umbilical cord mesenchymal stem cells (UC-MSCs) were obtained from fetuses with natural labor in Central People's Hospital of Zhanjiang. The experiment has been approved by Institutional Ethics Committee of Central People's Hospital of Zhanjiang (IIT-2022-057-01). Briefly, the fresh umbilical cord was cut off into clumps and were digested with 1 g/L collagenase II for 1 h. The digested clumps were then cultured in DMEM supplemented with 20% fetal bovine serum (FBS, Gibco, 12800), 100 U/mL penicillin, and 100 U/mL streptomycin at 37°C under 5% CO2. After 16 days, the clumps were removed carefully and cells were serially passaged after reaching 80−90% confluence. Cells are qualified for microbiology and virology tests. The population doublings were calculated with PD = log2 (Ne/N0), where N0 represents the number of seeds and Ne represents the number of counts. Cumulative PD numbers represent the sum of PDs from all previous passages, with cells derived from the primary culture considered PD = 0. CUT&Tag analysis was performed using the Hyperactive® Universal CUT&Tag Assay Kit for Illumina (Vazyme, TD903) as described by the manufacturers with some modifications. Briefly, nuclei were extracted from the 10,000 fresh cells, and added the ConA beads incubating at room temperature for 10 min. The mixed complexes were the incubated with H3K27ac and TEAD4 specific primary antibodies with IgG antibody as a control, and the next day, followed by the secondary antibody. A diluted pA-Tn5 adapter complex was then added to the above system and incubated at room temperature for 1 h. After extensive washing, system were subsequently sheared by adding 5 × TTBL. Subsequent proteinase K and DNA extract beads were used for obtaining eluted DNA. Finally, the sequencing libraries were established after 12 cycles of PCR amplification.