MinION sequencing; Direct RNA seq of activated primary B cells isolated from wild type mouse
Total RNA was isolated from cells with TRIzol reagent (Thermo Fisher Scientific, 15596) according to the manufacturer's instructions, dissolved in nuclease-free water and stored at -20°C. 100 µg of RNA was subjected to mRNA-enrichment with GST-eIF4EK119A protein, as described previously with some modifications (Bajak and Hagedorn, 2008).   Briefly, Glutathione-Sepharose 4B resin (GE Healthcare,) was incubated with GST-eIF4EK119A protein in sterile PBS (200 µl resin per 200 µg protein) for 1 hour at room temperature with rotation. Then the resin was washed 2 times with PBS and 3 times with buffer B (10 mM potassium phosphate buffer, pH 8.0, 100 mM KCl, 2 mM EDTA, 5% glycerol (Sigma), 0.005% Triton X-100 (Sigma), 6 mM DTT (AA Biotechnology), and 20 U/mL Ribolock RNase Inhibitor (Thermo Scientific). 100 µg of total RNA, previously denaturated during 10 min in 70°C, was mixed with the prepared resin and incubated for 1 h at room temperature on an immunoprecipitation rotor. Then, the resin was washed 3 times with buffer B, 2 times with buffer B supplemented with 0.5 mM GDP (Sigma) and 2 times with buffer B without GDP. RNA was eluted from the resign by acid phenol:chloroform extraction and precipitated using 100% ethanol (Merck), 3M sodium acetate and GlycoBlue coprecipitant (Invitrogen).   mRNA enrichment was followed by the ribodepletion of 2.5 µg RNA using a Ribo-Zero Kit (Illumina), according to the manufacturer's recommendations.   Nanopore direct RNA libraries were prepared from 500 ng of cap-enriched, rRNA-depleted mRNA with Direct RNA Sequencing Kit (ONT, SQK-RNA001). Instead of RNA CS, a 0.5 µl ERCC spike-in was added during RTA adapter ligation step and all remaining steps were performed according to the manufacturer's instructions.