GSM5203561: Riboseq_E14_2; Mus musculus; OTHER
Neocortex tissue was lysed on ice in 20 mM HEPES, 100 mM KCl, 7.5 mM MgCl2, pH 7.4, supplemented with 20 mM Dithiothreitol (DTT), 0.04 mM Spermine, 0.5 mM Spermidine, 1x Protease Inhibitor cOmplete EDTA-free (Roche, 05056489001), 0.3% v/v IGEPAL CA-630 detergent (Sigma, I8896) and clarified by centrifugation at 16100 xg for 5 min at 4 °C with a benchtop centrifuge. Samples were then measured for A260 ODU on a NanoDrop 1000 Spectrophotometer. For digestion of ribosome protected RNA fragments (RPFs), Ribo-seq samples were then mixed with 60U RNAse-T1 plus 96 ng RNAse-A per ODU, and incubated for 30 min at 25 °C, shaking at 400 rpm. To stop RNAse activity, 200 U of SUPERase-In RNAse inhibitor was then added. Riboseq sequencing library preparation: the RNA was first ligated to a 3' adapter 4N-RA3, and gel-purified using a 15 % denaturing urea-PAGE gel . Next the 5' adapter OR5-4N was ligated and gel purified. The RNA was reverse transcribed and PCR-amplified by Phusion High-Fidelity DNA polymerase (Thermo Fischer, F-530XL). The cDNA was visualized on a 2.5 % agarose gel, a ~150 bp sized fragment was excised and purified by Zymoclean Gel DNA Recovery kit (Zymo Research, D4007/D4008).