biostudies-arrayexpressYihang ShenHomo sapienshttps://www.ebi.ac.uk/biostudies/studies/E-MTAB-15387We have observed that the expression of IFIT1, IFIT2, IFIT3 and the other genes related interferon are all elevated by GLP1R knockdown whereas reduced by GLP1RA and 2GRA in multiple lineages differentiation systems of PDLSCs. It was reported that IFIT proteins could block the formation of 43S pre-initiation complex and 48S initial complex via interacting with eIF3E and eIF3C. RIP-seq was conducted to study the transcriptome-wide occupancies of IFIT1 and eIF3C in osteogenic PDLSCs treated with GLP1R knockdown compared to regular osteogenesis.biostudies-arrayexpressNucleic Acid Extraction - 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.Sample Collection - 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).Sequencing - The final libraries were quality-checked using a Bioanalyzer, and sequencing was performed on a NovaSeq 6000 platform with 150 bp paired-end reads.Library Construction - 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.OrganizationMINSEQE ScoreAssays and DataMAGE-TAB FilesData Transformation - Raw sequencing reads obtained from RIP-seq experiments were subjected to quality control using FastQC, and adapter trimming was performed with Cutadapt. High-quality reads were aligned to the reference genome (e.g., mm10 for mouse or hg38 for human) using STAR or HISAT2 with default parameters. PCR duplicates were removed to minimize amplification artifacts using SAMtools or Picard tools. Raw read counts for each gene or transcript were quantified using featureCounts or HTSeq-count. To normalize for differences in sequencing depth and sample composition, the trimmed mean of M-values (TMM) normalization method implemented in the edgeR package was applied. For comparison between immunoprecipitation (IP) and input samples, counts were scaled to counts per million (CPM) or reads per kilobase of transcript per million mapped reads (RPKM) as appropriate. Log2 transformation of normalized counts or log2 fold-change (IP/Input) was calculated to identify enriched transcripts. Additionally, variance-stabilizing transformation (VST) or regularized log transformation (rlog) from DESeq2 was applied for clustering, heatmap visualization, and principal component analysis (PCA). All normalization and transformation steps were performed using R and Bioconductor packages following best practices for RIP-seq data analysis.UnknownTranscriptomicsGenomicsIllumina NovaSeq 6000RIP-seqHomo sapiensYihang ShenfalseTranscriptome-wide occupancies of IFIT1 and eIF3C in osteogenic PDLSCs treated with GLP1R knockdownWe have observed that the expression of IFIT1, IFIT2, IFIT3 and the other genes related interferon are all elevated by GLP1R knockdown whereas reduced by GLP1RA and 2GRA in multiple lineages differentiation systems of PDLSCs. It was reported that IFIT proteins could block the formation of 43S pre-initiation complex and 48S initial complex via interacting with eIF3E and eIF3C. RIP-seq was conducted to study the transcriptome-wide occupancies of IFIT1 and eIF3C in osteogenic PDLSCs treated with GLP1R knockdown compared to regular osteogenesis.2025-07-06T00:00:00Z2025-07-22T17:01:49.328Z2025-07-22T17:01:49.328ZE-MTAB-15387ERP177068EFO_0002944EFO_0004170EFO_0005310EFO_0005518EFO_0003816EFO_0004184