Enrichment of m6A and G3BP1 on mRNA of bursa of Fabricius of Muscovy ducklings infected by HN10 NDRV
Enrichment of m6A and G3BP1 on mRNA of bursa of Fabricius of Muscovy ducklings infected by HN10 NDRV
Tissues containing at least 1×107 cells were harvested and resuspended in 2 ml of nuclear separation buffer (1.28 M sucrose, 40 mM Tris pH 7.5, 20 mM MgCl2, and 4% Triton X-100) and kept on ice for at least 30 min with frequent mixing. The pelleted nuclei were centrifuged at 14,000 rpm for 15 min and resuspended in wash buffer (150 mM KCl, 25 mM Tris (pH 7.4), 5 mM EDTA, 0.5 mM DTT, 0.5% NP40, 100 U/ml RNase inhibitor (Solarbio, Beijing, China), and 1× protease inhibitor cocktail (Solarbio)). For MeRIP, total RNA was isolated using TRIzol. Nuclei pullets or RNA were sheared via sonication at high power for 30 cycles of 5 s on and 30 s off. After that, the supernatant was incubated with 1 μg of antibodies against m6A (Cat. No. ab284130, Abcam) or G3BP1 (Cat. No. 13057-2-AP, Proteintech, Wuhan, Hubei, China) overnight and 40 μl of protein A/G beads (Thermo Fisher Scientific) for 2 h with gentle rotation at 4°C, while the remaining 10% was harvested as input. The pellet beads were centrifuged at 3,000 rpm for 3 min and washed three times. Both the input and pellet beads were purified by TRIzol, and reverse transcription was conducted using a HiScript III 1st Strand cDNA Synthesis Kit (Roche).