ena-STUDY-INTERNATIONAL INSTITUTE OF MOLECULAR AND CELL BIOLOGY IN WARSAW-20-10-2020-11:52:54:379-32
TENT5 cytoplasmic non-canonical poly(A) polymerases regulate the innate immune response in animals
In this study, we aimed to identify mRNAs that are polyadenylated by TENT5 non-canonical poly(A) polymerases in Caenorhabditis elegans and murine macrophages using genome-wide poly(A) tail profiling with Oxford Nanopore direct RNA sequencing. For C. elegans samples (CE), synchronized wild-type and tent-5(tm3504) mutant worms were grown on NGM plates seeded with E. coli HB101 at 25°C until they reached the L4 stage. Two independent replicate sample sets were prepared for each strain. For mice samples (BMDM), the primary bone-marrow derived macrophage (BMDM) cell cultures were established from the bone marrow monocytes from homozygous and heterozygous TENT5A/C knock-out (KO) littermate mice and cultured in IMDM medium (Invitrogen, 21980065) supplemented with macrophage colony-stimulating factor (M-CSF, Preprotech, 315-02) at 37°C in 5% CO2 as described previously (Graczyk et al., 2015).   Two replicate sample sets were prepared for homozygous (hom) and heterozygous (het) TENT5A/C KO littermates. RNA isolation from CE and BMDM samples was performed with Trizol reagent (Invitrogen, 15596) according to the manufacturer's instructions. 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).
In this study, we aimed to identify mRNAs that are polyadenylated by TENT5 non-canonical poly(A) polymerases in Caenorhabditis elegans and murine macrophages using genome-wide poly(A) tail profiling with Oxford Nanopore direct RNA sequencing. For C. elegans samples (CE), synchronized wild-type and tent-5(tm3504) mutant worms were grown on NGM plates seeded with E. coli HB101 at 25°C until they reached the L4 stage. Two independent replicate sample sets were prepared for each strain. For mice samples (BMDM), the primary bone-marrow derived macrophage (BMDM) cell cultures were established from the bone marrow monocytes from homozygous and heterozygous TENT5A/C knock-out (KO) littermate mice and cultured in IMDM medium (Invitrogen, 21980065) supplemented with macrophage colony-stimulating factor (M-CSF, Preprotech, 315-02) at 37°C in 5% CO2 as described previously (Graczyk et al., 2015).   Two replicate sample sets were prepared for homozygous (hom) and heterozygous (het) TENT5A/C KO littermates. RNA isolation from CE and BMDM samples was performed with Trizol reagent (Invitrogen, 15596) according to the manufacturer's instructions. 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).