Genome-wide occupancies of multiple transcription activators in PDLSCs induced by GIPRA
Genome-wide occupancies of multiple transcription activators in PDLSCs induced by GIPRA
Human periodontal ligament (PDL) tissues were obtained from five healthy orthodontic donors with informed consent. Immediately after extraction, the teeth were rinsed thoroughly with sterile phosphate-buffered saline (PBS) containing 100 U/mL penicillin and 100 μg/mL streptomycin (Cat. No. 15140122, Thermo Fisher Scientific, USA) to remove blood and debris. PDL tissues were carefully scraped from the middle third of the root surface using a sterile scalpel under aseptic conditions to minimize contamination from gingival and pulp tissues. PDLSCs were primarily cultured within α-MEM supplemented with 15% fetal bovine serum (Thermo Fisher Scientific), and 1 × GlutaMAX (Cat. No. 35050061, Thermo Fisher Scientific).The culture medium for inflammatory PDLSCs were additionally supplemented with 10 ng/mL recombinant human FGF-2 (Cat. No. ab9596, Abcam, USA). 5 × 10^7 PDLSCs were cross-linked with 1% formaldehyde at room temperature for 10 min, and the reaction was quenched with 0.125 M glycine. Chromatin was isolated and sheared to an average size of 200-500 bp under the condition (Power, high intensity; Cycle, 30 s On/30 s Off for 30 cycles; Temperature, 4 °C) using a Bioruptor Pico Sonication Device (Cat. No. B01080010, Hologic Diagenode, Belgium). Immunoprecipitation was carried out overnight at 4 °C using 1 μg IP grade antibodies against p-CREB1 (Cat. No. 81871-1-RR, Proteintech), p-ERK1/2 (Thr202/Tyr204), p-STAT3 (Ser727) (same with Western blot), TCF4 (Cat. No. 22337-1-AP, Proteintech). Next day, the immune complex were added 30 μL Pierce Protein A/G magnetic beads (Cat. No. 88802, Thermo Fisher Scientific) slurry after vortex, and washed, eluted, and reverse cross-linked, followed by DNA purification using a VAHTS Universal Plus DNA Library Prep Kit V4 (Cat. No. ND801-01, Vazyme). ChIP and input DNA were used to prepare sequencing libraries with a NEBNext Ultra II RNA Library Prep Kit for Illumina