title: MinION sequencing; Nanopore direct RNA sequencing of M. musculus bone marrow derived macrophages TENT5A -/- TENT5C -/- , biological replicate 2
library_construction_protocol: RNA isolation from 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). Nanopore direct-RNA libraries were prepared with Direct RNA Sequencing Kit (Oxford Nanopore Technologies, SQK-RNA002) from 3 µg of cap-enriched mRNA mixed with 150 ng of Saccharomyces cerevisiae oligo(dT)-enriched mRNA to optimize sequencing efficiency. Sequencing was performed with MinION device (Oxford Nanopore Technologies, Flow cell type FLO-MIN106; RevD) and basecalled with Guppy (Oxford Nanopore Technologies)