ena-STUDY-INTERNATIONAL INSTITUTE OF MOLECULAR AND CELL BIOLOGY IN WARSAW-11-08-2020-14:30:15:044-2246
Cytoplasmic polyadenylation by TENT5A is required for proper bone formation.
The goal of this study was to assess changes in the mineralizing osteoblasts transcriptome upon TENT5A knockout in mice. Neonatal calvarial and adult long-bones osteoblasts were isolated from TENT5A KO or WT mice. Adult osteoblasts were passaged 3 times before harvesting. Neonatal osteoblasts were passage once, after reaching the confluency cells were either harvested (for D0 timepoint) or medium was changed to differentiation medium (MEM alpha supplemented with 10% FBS, 50 µg/ml sodium ascorbate (Sigma) and 10 mM ßglycerophosphate (Roth) and cultured for 14 days with medium change 2 times per week and harvested (for D14 timepoint). Total RNA was isolated with TRIzol (Thermo Fisher Scientific) according to the manufacturer's instructions, dissolved in nuclease-free water and stored at -80°C. The cap-enriched mRNA was prepared from 100?µg of total RNA with GST-eIF4EK119A protein and glutathione sepharose 4B (GE Healthcare), as described previously (Bilska et al., 2020).  Nanopore Direct RNA libraries were prepared using Direct RNA Sequencing KIT SQK-RNA002 (ONT) according to the manufacturer's instructions, from 5 ug of murine cap-enriched mRNA and, for better sequencing efficiency, mixed with the 100-150?ng of Saccharomyces cerevisiae oligo(dT)-enriched mRNA. Sequencing was performed with the MinION device and basecalled using Guppy (ONT).
The goal of this study was to assess changes in the mineralizing osteoblasts transcriptome upon TENT5A knockout in mice. Neonatal calvarial and adult long-bones osteoblasts were isolated from TENT5A KO or WT mice. Adult osteoblasts were passaged 3 times before harvesting. Neonatal osteoblasts were passage once, after reaching the confluency cells were either harvested (for D0 timepoint) or medium was changed to differentiation medium (MEM alpha supplemented with 10% FBS, 50 µg/ml sodium ascorbate (Sigma) and 10 mM ßglycerophosphate (Roth) and cultured for 14 days with medium change 2 times per week and harvested (for D14 timepoint). Total RNA was isolated with TRIzol (Thermo Fisher Scientific) according to the manufacturer's instructions, dissolved in nuclease-free water and stored at -80°C. The cap-enriched mRNA was prepared from 100?µg of total RNA with GST-eIF4EK119A protein and glutathione sepharose 4B (GE Healthcare), as described previously (Bilska et al., 2020).  Nanopore Direct RNA libraries were prepared using Direct RNA Sequencing KIT SQK-RNA002 (ONT) according to the manufacturer's instructions, from 5 ug of murine cap-enriched mRNA and, for better sequencing efficiency, mixed with the 100-150?ng of Saccharomyces cerevisiae oligo(dT)-enriched mRNA. Sequencing was performed with the MinION device and basecalled using Guppy (ONT).