GSM6381793: blastocyst_Ribo-seq_rep2; Mus musculus; OTHER
After removing the zona pellucida, embryonic cells were washed with PBS and incubated in lysis buffer. Samples were lysed in 310 μl ice-cold lysis buffer (20 mM Tris-HCL ph7.4; 150 mM NaCl; 5 mM MgCl2; 1mM DTT; 100 μg/ml Cycloheximide; 1% Triton; 25 U/ml Turbo DNase Turbo DNase (Ambion, AM2239)) for 10 min. The lysate was clarified for 10 min at 20,000g at 4°C, then treated with 1 μl RNase I 100 U/ml (Ambion, AM2295) and incubated at room temperature for 45 min with gentle mixing. The digested extracts were stopped by the addition of 10 μl SUPERas•In (Ambion, AM2696) and were overlaid onto a 700 μl sucrose cushion of 1M sucrose in 20mM total Tris-HCL pH7.4; 150 mM NaCl; 5 mM MgCl2; 1 mM DTT; 100 μg/ml Cycloheximide; 20 U/ml SUPERas•In (Ambion, AM2696) for 4 hr at 76,400 rpm in a MLA-150 rotor in a Beckman Optima MAX-XP ultracentrifuge. The supernatant was removed and the pellet was resuspended in 50 μl pellet buffer (10 mM Tris (pH 7.5); 1% SDS). RNA was extracted from pellet buffer using TRIzol (life tech, 15596018), chloroform and aqueous phase was precipitated by at least one volume of isopropanol and 20 μg glycogen. Precipitation was carried out at -20°C overnight and RNA was then pelleted by centrifugation for 40 min at 12,000g, 4°C. RNA was resuspended in 6 μl Nuclease-free water and mix with 6 μl Gel Loading Buffer II (ThermoFisher, AM8554G).