Transcriptome-wide occupancies of IFIT1 and eIF3C in osteogenic PDLSCs treated with GLP1R knockdown
Transcriptome-wide occupancies of IFIT1 and eIF3C in osteogenic PDLSCs treated with GLP1R knockdown
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). 1 × 10^9 PDLSCs were lysed in RIP lysis buffer (150 mM KCl, 25 mM Tris-HCl, pH 7.4, 0.5 mM DTT, 0.5% NP-40, 1 mM PMSF, supplemented with RNase inhibitor (Cat. No. N8080119, Thermo Fisher Scientific) and protease inhibitor cocktail). The lysates were cleared by centrifugation and incubated with Protein A/G magnetic beads conjugated to antibodies against IFIT1 or eIF3C at 4°C overnight with gentle rotation. After incubation, beads were washed thoroughly with high-salt wash buffer (500 mM NaCl, 25 mM Tris-HCl, pH 7.4, 0.5% NP-40) to remove nonspecific interactions.RNA-protein complexes were then eluted from the beads, followed by proteinase K digestion to remove proteins. The immunoprecipitated RNAs were extracted using TRIzol Plus RNA Isolation Kit, and RNA quality was assessed.  Ribosomal RNA (rRNA) was depleted from the RNA samples using the KAPA RiboErase Kit (Cat. No. 07962304001, Roche KAPA, Switzerland) according to the manufacturer's protocol. The rRNA-depleted RNA was then fragmented by heat treatment to generate RNA fragments approximately 200–300 nucleotides in length. Strand-specific cDNA synthesis was subsequently performed using a strand synthesis master mix. Following cDNA synthesis, the cDNA fragments were end-repaired and subjected to 3' adenylation. Sequencing adapters were then ligated to the cDNA fragments. After adapter ligation, PCR amplification was conducted to enrich the library, followed by size selection of DNA fragments to obtain the desired library size.