Fhit study pIND-Fhit vector replicate 1 RIBO-Seq
Cells were seeded, cultured, and induced as indicated above. Cycloheximide (CHX, 100 μg/ml) was added to the medium for 10 min at 37°C. Cells were then washed twice with PBS+CHX (100 μg/ml), scraped, and pelleted by centrifugation at 2500 xg for 5 min at 4°C. Ribosome profiling was performed essentially as outlined in [1] with key alterations detailed below. Cell pellets were lysed in 5 pellet volumes of ice cold polysome lysis buffer (20 mM Tris pH 7.5, 150 mM NaCl, 5 mM MgCl2, 1 mM DTT, 150 μg/ml CHX, 1% Triton X-100, and 0.1 U/μl DNase 1), incubated on ice for 10 mins with mild agitation every 2 minutes. Cellular debris was pelleted by centrifugation (16,100 xg) for 10 mins at 4°C. 10% of the supernatant was reserved and RNA was harvested as above for sample-matched total cytoplasmic RNA-seq analyses. The remaining material was digested with RNase I (Ambion, 2.5 U/μl) for 45 minutes at 25°C. Superasin (Life technologies, 5 μl per 100 μl of lysate) was added to stop the digestion, samples were vortexed and place on ice. Digested samples were loaded into thick-walled ultracentrifuge tubes and a 1 ml sucrose cushion was underlayed. Samples were centrifuged in a Sorvall Discovery M120 SE ultracentrifuge using a S-55A rotor at 54,000 RPM for 4 hours at 4°C. Supernatants were discarded and pelleted RNA was harvested as described in [1], but samples were sequentially eluted off the miRNeasy column using 40 μl and 35 μl of water. At this point, the two elutions were pooled and two additional ribosomal RNA removal steps were added to decrease ribosomal RNA contamination thereby increasing the proportion of ribosome protected fragments (RPFs) in the final libraries. First, we generated a pool of biotinylated oligonucleotides to hybridize directly to and deplete rRNA sequences that were often incorporated into libraries. The amount of each biotinylated oligo in the pool (17 μl per sample) is dependent upon the prevalence of the contaminating rRNA sequence in previous ribosome profiling library preparations. 10 μl of 20x SSC and 17 μl of the premixed depletion pool was added to the RPFs harvested earlier. RNA secondary structures were denatured by incubation at 80°C for 1 min in a thermocycler. Samples were then gradually cooled (-1°C every 20 sec) to 70°C and held at 70°C for 1 minute. Samples were then gradually cooled (-1°C every 30 sec) to 37°C. After cooling, pre-equilibrated MyOne Streptavidin C1 para-magnetic beads (100 μl of slurry per sample, Life Technologies) were added to each sample and incubated with mixing (350 RPM) in a thermomixer at 37°C for 15 minutes. Tubes were then immediately transferred to a magnetic stand for ~5 minutes and the supernatant (~200 μl) was transferred to a fresh tube. Each sample was split into two tubes and precipitated with the addition of 284 μl H2O, 4 μl of Glycoblue, and 970 μl of 100% EtOH followed by incubation at -20°C overnight. Remaining RPFs were pelleted by a 35 min centrifugation at 16,100 xg at 4°C. Pellets were rinsed with 70% EtOH and recovered RPFs were resuspended in 28 μl of Tris pH 8.0 and further purified using Ribo-Zero Gold according to the manufacturer's (Illumina) instructions for 1 μg of input RNA. Importantly, the final 50°C incubation step was omitted from the Ribo-Zero Gold protocol and the purified RNAs were resuspended in 5 μl of Tris pH 8.0 . The remainder of the protocol was performed as described in [1] except different biotinylated oligonucleotides were used for subtractive hybridization of circularized ribosome-specific cDNAs and new barcode oligos were substituted for those listed in [1]. References: [1] Ingolia NT, Brar GA, Rouskin S, McGeachy AM, Weissman JS. The ribosome profiling strategy for monitoring translation in vivo by deep sequencing of ribosome-protected mRNA fragments. Nat Protoc. 2012;7:1534-1550.