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Ingolia et al., 2012 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.

Cells were treated with 100 μg/ml cycloheximide in mESC media for 1 min prior to harvest. 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 MgCl, 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) and incubated for 30 min at 4°C with occasional vortexing. 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 the nuclei and mitochondria. 250 μL of lysate (with RNA concentration of ∼1 μg/μL) was then treated with RNase A/T1 mix (0.5 mg RNase A (Ambion, 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). 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, the pellets were re-suspended in TRIzol for RNA extraction. Two biological replicates were performed for each type of Ribo-Seq and all Ribo-Seq libraries were prepared as described before (). Details are described as below. First, ribosome protected fragments extracted from TRIzol were run on a 15% TBE-Urea polyacrylamide gel and size selected between 28-nt and 34-nt as marked by RNA oligonucleotides oNTI199 and oNTI265 ( Table S5 ). Gel slices were crushed with a razor blade and incubated overnight at room temperature in 400 μL of RNA extraction buffer (300 mM NaOAc pH 5.5, 1 mM EDTA, 0.25% SDS). RNA was then extracted with acid-phenol:chloform and precipitated with isopropanol. RNA were then 3′ dephosphorylated with 1 μL of T4 Polynucleotide Kinase (PNK) (NEB, M0201S) in 50 μL of total volume for 1 hr at 37°C. This was followed by acid-phenol:chloform extraction and isopropanol precipitation. 3′ dephosphorylated RNAs were then incubated with 1.5 μL of 0.5 μg/μL Universal miRNA Cloning Linker (NEB, S1315S) and 1 μL T4 RNA Ligase 2, truncated (NEB, M0242S) in 20 μL of total volume for 2.5 hr at room temperature. Samples were then purified by acid-phenol:chloform extraction and isopropanol precipitation. 3′ ligated ribosome protected fragments were subsequently size selected on 10% TBE-Urea polyacrylamide gels and purified. For subsequent reverse transcription, purified 3′ ligated products were incubated with 2 μL of 1.25 μM RT primer ( Table S5 ) and denatured for 2 min at 80°C. Reverse transcription was performed with SuperScript III (Invitrogen, 18080-044) in a 20 μL of total volume (30 min, 48°C). RNA was then hydrolyzed by adding 2.2 μL of 1M NaOH and incubated for 20 min at 98°C. cDNAs were purified by isopropanol precipitation. RT products were size selected on 10% TBE Urea polyacrylamide gels. Gel slices were crushed with a razor blade and incubated overnight at room temperature in 400 μL of DNA extraction buffer (300 mM NaCl, 10 mM Tris-HCl pH 8, 1 mM EDTA, 0.1% SDS). DNA was precipitated with isopropanol overnight at −80°C. DNA was then circularized with CircLigase (Illumina, CL4115K) in a 20 μL volume at 60°C for 12 hr. To deplete rRNAs, circularized DNA sample was incubated with 4 μL of biotinylated oligonucleotide pool (reverse complements to pieces of rRNA sequences, 10 μM for each oligo; see Table S5 ) in 40 μL with 0.5xSSC (75 mM NaCl, 7.5 mM sodium citrate). Samples were denatured at 100°C for 90 s and slowly cooled to 37°C (0.1°C/sec), followed by incubation at 37°C for 15 min. For each sample, 100 μL of MyOne Streptavidin C1 DynaBeads (Invitrogen, 65001) was washed, re-suspended in 40 μL of 2x bind/wash buffer (2M NaCl, 1mM EDTA, 5mM Tris (pH 7.5), and 0.2% Triton X-100) and mixed with the sample. The sample was then incubate at 37°C for 15 min with mixing (1,000 rpm). Supernatants were collected and precipitated by adding 2 μL of Glycogen (Ambion, AM9510), 6 μL of 5 M NaCl and 150 μL of isopropanol. Purified cDNA were dissolved in 10 μL of ultrapure water. 1 μL of cDNA was used as template for PCR amplification with Phusion High-Fidelity DNA Polymerase (Thermo Fisher, F530S) for 10-11 amplification cycles, with primers listed in Table S5 . PCR product was purified from 8% TBE polyacrylamide gels. The quality and concentration of DNA were measured with the Agilent 2100 Bioanalyzer (High-Sensitivity DNA) by the Stanford Protein and Nucleic Acid Facility. Finally, libraries were sequenced on the Illumina NextSeq 500 sequencer (1x75 nt) by the Stanford Functional Genomics Facility.