title: GSM2579681: RPL22-Ribo-Seq (RPL22-3xHA cells)_rep1; Mus musculus; OTHER
library_construction_protocol: 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). 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, 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. 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. For RPL22-Ribo-Seq, ribosomes containing RPL22 were enriched by IP by incubating with anti-HA antibody (ab9110) on a head-over-tail rotator at 4 °C for 4 hrs and and then Dynabeads Protein A (Life Technologies, 10001D) at 4 °C for 1 hr. 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. Polysome profiling was performed comparing the control siRNA and Rpl10a knockdown. After polysome fractionation, RNA was extracted using Acid-Phenol:Chloroform, pH 4.5 (with IAA, 125:24:1) (Ambion, AM9722). We collected RNA from combined light fractions containing the free RNPs, 40S/60S ribosomal free subunits, 80S/monosome as well as light polysomes (2-3 ribosomes on a mRNA molecule), and combined heavy polysome fractions containing the most actively translating ribosomes (≥4 ribosomes along a mRNA molecule).
attributes.tag: GEO Accession
attributes.value: GSM2579681