GSM5342779: NSP2-H1299-2_ribo; Homo sapiens; OTHER
Ribosome Profiling: Three million HEK293T or H1299 cells were plated in a 10 cm2 flask followed by transfection to express SARS-CoV-2 NSP1 and NSP2 (see above). Untransfected and transfected cells (~8 million) were washed twice with 10 mL of ice-cold PBS. The plates were immediately placed on ice and 400 μL of lysis buffer (Tris-Cl, pH 7.4, 150 mM NaCl, 5 mM MgCl2, 1 mM DTT, 100 µg/mL Cycloheximide, 1% Triton-X) was added to each plate, cells were scraped and transferred to 1.5 ml tubes. Cells were lysed on ice by pipetting up and down ~5 times every five minutes for a total of 10 min. All experiments were done in triplicate. The lysate was clarified by centrifugation at 160 x g for 10 min at 4°C. Ten percent of the clarified lysate by volume was separated, mixed with 700 μl QIAzol and stored at -20°C for RNA-Seq experiments (see below). The rest of the supernatant was immediately processed for ribosome profiling. Briefly, 7 μL of RNaseI (Invitrogen AM2249) was added to the clarified lysates and digestion was done for 1 h at 4 °C. The digestions were stopped with ribonucleoside vanadyl complex (NEB S1402S) at a final concentration of 20 mM. Digested lysates were layered on a 1 M sucrose cushion (Tris, pH 7.4, NaCl 150 mM, MgCl 2 5 mM, DTT 1 mM, 1 M sucrose) and the ribosomes were pelleted by centrifugation in a SW 41 Ti rotor (Beckman Coulter) at 38K rpm and 4°C for 2.5 h. RNA was extracted from the ribosome pellets with the addition of 700 μl QIAzol followed by chloroform and ethanol precipitation. RNA isolated from the pellets were size-selected by running 5 µg of each sample in a 15% polyacrylamide TBEUREA gel. The 21-34 nt RNA fragments were excised and extracted by crushing the gel fragment in 400 µL of RNA extraction buffer (NaOAc [pH 5.5] 300 mM, EDTA 1 mM, SDS 0.25% v/v) followed by a 30 min incubation on dry ice and an overnight incubation at room temperature. The sample was passed through a Spin X filter (Corning 8160) and the flow through was ethanol precipitated in the presence of MgCl2 5 mM and 1 µL of Glycoblue (Invitrogen AM9516). The RNA pellet was resuspended in 10 µL of RNase-free water and immediately processed for library preparation. Ribosome profiling libraries were prepared with the D-Plex Small RNA-Seq Kit (Diagenode). This method incorporates a 3′ end dephosphorylation step, 5′ unique molecular identifiers (UMI) and a template switching reverse transcriptase that improves the quality of our libraries. Briefly, 25 ng of size-selected ribosome footprints were prepared following the manufacturer's instructions with some modifications. The cDNA was amplified for 9 cycles and the resulting DNA libraries were pooled in equimolar amounts (~425 nM each). The library pool was cleaned with the AMPure XP beads (Beckman Coulter A63880) and eluted with 30 µL of RNase-free water. To enrich for ~30 bp fragments in the libraries, 3 µg of the cleaned libraries were size-selected in a 3% agarose precast-gel (Sage Science) with the BluePippin system (Sage Science) using 171-203 nt range with tight settings. The resulting size-selected libraries were analyzed with the Agilent High Sensitivity DNA Kit (Agilent) and sequenced with the NovaSeq 6000 S1 SE 75 (Illumina).
GEO
GSM5342779