GSM1891627: RPS25-Ribo-Seq (Rps25-3xFLAG cells)_rep2; Mus musculus; OTHER
The media was then aspirated and cells were scraped from the plate in cold PBS with 100 μg/ml cycloheximide. After centrifugation, the cell pellet was re-suspended in cold lysis buffer (20 mM Tris pH 7.5, 150 mM NaCl, 15 mM MgCl2, 100 μg/ml cycloheximide, 1 mM DTT, 0.5% Triton X-100, 0.1 mg/ml heparin, 8% glycerol, 20 U/ml TURBO™ DNase, 1x Combined Protease and Phosphatase Inhibitor (Thermo 78443)) and incubated for 30 min at 4°C with occasional vortex. The lysate was clarified by sequential centrifugation for 5 min at 1,800 x g and 10,000 x g at 4°C to remove nucleus and mitochondria. A 250 μl of lysate (with RNA concentration of ~ 1 μg/μl) was then treated with RNase A/T1 mix (0.5 mg RNase A (Life Technologies AM2272) and 1,000 U RNase T1 (Life Technologies 2280)) for 30 min at room temperature to digest mRNAs not protected by the ribosome. The digestion was stopped by adding 4.5 μl of SUPERase-In™ RNase Inhibitor (20 U/μl, Ambion AM2696). For the total Ribo-Seq, lysate was then loaded onto a 1 M sucrose cushion. Ribosomes were pelleted by centrifugation at 70, 000 rpm for 4 hr at 4°C. After a brief wash, pellet were re-suspended in TRIzol® for RNA extraction. For the RPL10A-Ribo-Seq and RPS25-Ribo-Seq, ribosomes containing RPS25 or RPL10A were enriched by IP using anti-FLAG® M2 magnetic beads (Sigma) on a head-over-tail rotator at 4 °C for 2 hrs. Beads were washed 5 min for 3 times in washing buffer (20 mM Tris pH 7.5, 200 mM NaCl, 15 mM MgCl2, 100 μg/ml cycloheximide, 0.5% Triton X-100). After the final wash, liquid was aspirated and immunoprecipitated ribosomes were eluted in TRIzol® for RNA extraction. All Ribo-Seq libraries were prepared as described in Ingolia, N. T., Brar, G. A., Rouskin, S., McGeachy, A. M. & Weissman, J. S. The ribosome profiling strategy for monitoring translation in vivo by deep sequencing of ribosome-protected mRNA fragments. Nat Protoc 7, 1534–1550 (2012)