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Briefly, embryos were lysed in 310 μL lysis buffer (20 mM Tris-HCl pH7.4, 150 mM NaCl, 5 mM MgCl2, 1mM DTT, 100 μg/mL CHX, 1% Triton; 25 U/mL Turbo DNase (Ambion, AM2239)) for 10 min. Then the lysate underwent centrifugation for 10 min at 17,000 g at 4 °C, followed by the addition of 1 μL RNase I 100 U/mL (Ambion; AM2295) and incubation on thermomixer at 22 °C with agitation at 1000 rpm for 45 min. The digestion process was halted by the addition of 3 μL of SUPERase In (Ambion; AM2696), and then layered onto a 700 μL sucrose cushion containing 1M sucrose in buffer (20 mM Tris-HCl pH 7.4, 150 mM NaCl, 5 mM MgCl2, 1 mM DTT, 100 μg/mL CHX, and 20 U/mL SUPERase In). Following centrifugation at 260,000 g for 4 h at 4 °C using an MLA-150 rotor within a Beckman Optima MAX-XP ultracentrifuge, the resultant pellet was resuspended in 50 μL of pellet buffer (10 mM Tris pH 7.5, 1% SDS). Ribosome-protected fragments (RPFs) were extracted from the pellet buffer using TRIzol according to the manufacturer’s instructions, with the addition of glycogen during RNA precipitation. RNA was resuspended in 6 μl Nuclease-free water and mix with 6 μl Gel Loading Buffer II (ThermoFisher, AM8546G). |
Ribo-seq |
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other |
PAIRED
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