Pmscv mlin28a

Human embryonic kidney 293T cells were cultured in DMEM with 10% FBS at 37°C and 5% CO₂. Ready-to-use packaging plasmid mix (Cellectar, Florham Park, NJ, USA) and ExGen 500 (Euromedex, Souffelweyersheim, France) transfection reagents were used for plasmid transfection. Plasmids (2 μg) for Lin-28a (pMSCV-mLin28A, Addgene plasmid no. 26357) and HMGA2 (pBabe zeo HMGA2, Addgene plasmid no. 17411; pLKO.shHmga2, Addgene plasmid no. 32399) or empty vector (CMV500 empty vector, Addgene plasmid no. 33348) were transfected into cells. Viral particles were obtained and stored at −80°C. Chondrocytes at 50 to 60% confluency were infected with viral particles (1 ml) and Polybrene (Sigma-Aldrich, Darmstadt, Germany) at 4 μg/ml for 48 hours. During culture, cells were maintained in a humidified incubator containing 5% CO₂ at 37°C and 3% O₂.

RNA-seq was performed in duplicate for the following conditions: primary murine chondrocytes were transduced for control conditions with empty vector (CMV500 empty vector; Addgene plasmid no. 33348) and for Lin28 overexpression with pMSCV-mLin28A (Addgene plasmid no. 26357). Total RNA was extracted and transferred for sequencing to IntegraGen SA Co. (Evry, France). Libraries were prepared with the NEBNext Ultra II directional RNA library prep kit for the Illumina protocol according to the supplier’s recommendations. Briefly, the successive stages of this protocol are the purification of PolyA containing mRNA molecules using poly-T oligo attached magnetic beads from 1 μg of total RNA (with the magnetic mRNA isolation kit from New England Biolabs), fragmentation using divalent cations under elevated temperature to obtain approximately 300–base pair (bp) pieces, double-strand cDNA synthesis, and, last, Illumina adapter ligation and cDNA library amplification by PCR for sequencing. Sequencing then involves paired-end 100-bp reads with Illumina NovaSeq. Image analysis and base calling involved using Illumina Real-Time Analysis (3.4.4) with default parameters.