Illumina HiSeq 2000 sequencing; Ribosome profiling upon overexpression of the RNA-binding protein Unkempt
Ribosome profiling upon overexpression of the RNA-binding protein Unkempt
HeLa cells were grown in Dulbecco's Modified Eagle Medium (DMEM) supplemented with 10% fetal bovine serum, 100 U/mL penicillin, and 100 micro-g/mL streptomycin at 37  degrees C and 5% CO2. To create Dox-inducible cell lines, we first transduced HeLa cells with MSCV-rtTA3-IRES-EcoR-PGK-Neo (pRIEN), a retrovirus we constructed in the MSCV backbone (Clontech) to co-express an improved reverse transactivator (rtTA3), the ecotropic receptor, and a neomycin resistance marker. Following G418 selection, stably rtTA3-expressing cells were subsequently infected ecotropically with a second retrovirus expressing a puromycin resistance gene and a TREtight-driven transcript encoding GFP alone (pTt-IGPP) or GFP and either full-length mouse or zebrafish Unkempt protein, or any of the mouse Unkempt-deletion mutants (pTtight-X-IGPP, where X is an ortholog or mutant of Unkempt), which we constructed in a self-inactivating retroviral backbone (pQCXIX, Clontech). To induce transgene expression, double-selected cells were treated with doxycycline (Sigma-Aldrich) at 1 micro-g/ml. To determine the effect of Unkempt on the rate of protein synthesis, we performed ribosome profiling experiment on inducible HeLa cells essentially as described (Ingolia et al., Nature Protocols, 2012), but using the linker and the primers as for the iCLIP experiments (Konig et al., JoVE, 2011). GFP-only or GFP and Unkempt-inducible Hela cells were grown in 15 cm dishes and harvested at 70% confluency. Prior to harvest, cells were treated with Dox for 18 hour to induce the expression of transgenes. Cells were then washed in ice-cold PBS, lysed for 10 minutes on ice in lysis buffer, triturated by passing twice through a syringe fitted with a 26-gauge needle, and spun at 20,000 xg for 10 minutes at 4  degrees C. Cell lysate was digested with RNase I for 45 mintues at room temperature followed by the addition of SUPERase IN to stop nuclease digestion. The lysate was underlayed with 1 M sucrose and spun in a TLA 100.3 rotor at 70,000 rpm for 4 hours at 4  degrees C. Ribosomal pellet was resuspended in Trizol reagent (Life Technologies) and RNA extracted according to the manufacturer's instructions. The extracted RNA was size-selected by denaturing PAGE, retaining only fragments between 26 and 34 nts, and 3' end-dephosphorylated with T4 PNK for 20 minutes at 37  degrees C. Dephosphorylated RNA was precipitated, resuspended, and ligated to a pre-adenylated linker (L3-App) as described for the iCLIP procedure (Konig et al., JoVE, 2011). The ligation mix was precipitated, resuspended, and the ligation products (between 47 and 55 nts) were purified by denaturing PAGE. Reverse transcription (RT) was carried out using barcoded Rclip primers used for the iCLIP experiments (Konig et al., JoVE, 2011) by resuspending the ligation products in 8.5 micro-l water, 0.5 ul Rclip primer (100 micro-M; Rclip primer with a different barcode was used for each sample), and 1 micro-l dNTP (10 mM), with SuperScript III Reverse Transcriptase (Life Technologies) according to the manufacturer's instructions, including the treatment with RNase H. The reaction mix was precipitated and the produced cDNA (between 79 and 87 nts) was purified by denaturing PAGE. RT products were circularized, cut, and precipitated as described for the iCLIP procedure (Konig et al., JoVE, 2011). PCR amplification (12-15 cycles) was carried out using P5/P3 Solexa primers and Accuprime Supermix 1 enzyme (Life Technologies). The prepared cDNA libraries were purified using the Agencourt AMPure XP system (Beckman Coulter).
Experimental Factor: phenotype
Doxycycline-induced GFP expression control