| Library |
| Library name |
Construction protocol |
Strategy |
Source |
Selection |
Layout |
|
Cultured cells were washed once with PBS and then subjected to RNA purification with TRIzol (ThermoFisher). Purified total RNAs were first treated with Turbo DNase (ThermoFisher) and then depleted the rRNAs with the Ribo-off rRNA Depletion Kit (Human/Mouse/Rat) (Vazyme) following the manufacturer’s instructions. Then, the purified ribominus RNAs were fragmented into a size of around 100 nt using RNA fragmentation reagents (ThermoFisher, AM8740). About 100 ng of RNAs were mixed with 5 μg of anti-m6A antibody (Synaptic Systems, 202003) and 20 μL Dynabeads Protein A (Invitrogen, 1001D) in 500 μL IPP buffer (150 mM NaCl, 10 mM Tris-HCl, pH 7.4, 0.1% NP-40) supplemented with 10 U RNase Inhibitor (Beyotime) and incubated at 4 °C for 4 h with rotation. After extensive washing with IPP buffer, high-salt wash buffer (500 mM NaCl, 10 mM Tris-HCl, pH 7.4, 0.1% NP-40), and low-salt wash buffer (50 mM NaCl, 10 mM Tris-HCl, pH 7.4, 0.1% NP-40), the RNA fragments were eluted from the beads by proteinase K (Roche, 3115836001) digestion at 55 °C for 1 h, phenol-chloroform extraction and ethanol precipitation. RNAs were recovered and subjected to library preparation using the KAPA RNA HyperPrep kit (KAPA Biosystems, KK8541). |
RIP-Seq |
TRANSCRIPTOMIC |
size fractionation |
PAIRED
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