title: m6A_NC_input_1
organism: Anas platyrhynchos
description: Protocols: 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).
attributes.tag: ENA first public
attributes.value: 2024-11-15
attributes.tag: INSDC center name
attributes.value: State Key Laboratory for Managing Biotic and Chemical Threats to the Quality and Safety of Agro-products, Institute of Animal Husbandry and Veterinary Sciences, Zhejiang Academy of Agricultural Sciences
attributes.tag: INSDC status
attributes.value: public
attributes.tag: Submitter Id
attributes.value: E-MTAB-14565:m6A_NC_input_1
attributes.tag: age
attributes.value: 1
attributes.tag: broker name
attributes.value: ArrayExpress
attributes.tag: collection date
attributes.value: not collected
attributes.tag: common name
attributes.value: mallard
attributes.tag: developmental stage
attributes.value: neonatal
attributes.tag: disease
attributes.value: Negative control
attributes.tag: genotype
attributes.value: wild type genotype
attributes.tag: geographic location (country and/or sea)
attributes.value: not collected
attributes.tag: isolate
attributes.value: not applicable
attributes.tag: organism part
attributes.value: bursa of Fabricius
attributes.tag: sample name
attributes.value: E-MTAB-14565:m6A_NC_input_1
attributes.tag: scientific_name
attributes.value: Anas platyrhynchos
attributes.tag: sex
attributes.value: male
attributes.tag: strain
attributes.value: muscovy ducklings