GSM8328754: Hv ribosome footprints, digested with MNR, biol rep 1; Haloferax volcanii; OTHER
As previously described (Gelsinger et al. 2022), cells were harvested by dispensing culture into liquid nitrogen, forming small pellets. Batches of 50 g of pellets were supplemented with 100 μg/mL anisomycin (Sigma-Aldrich, #A9789) and pulverized in a cryomill (6870 Large Freezer/Mill (Spex)) for 8 cycles (1 min grinding at 10 Hz, 1 min cooling). Cell lysates were thawed at RT and pre-cleared at 10,000 x g for 15 min at 4°C. Ribosomes were pelleted from pre-cleared lysate over a 1 M sucrose cushion [sucrose dissolved in 1× lysis buffer (3.4 M KCl, 100 mM MgCl2, 50 mM CaCl2, 10 mM Tris-HCl pH 7.5)] in an ultracentrifuge with a Type 45 Ti rotor (Beckman Coulter) at 40,000 rpm (185,511 x g) for 3 h at 4°C. Ribosome pellets were resuspended in 200 μL 1x lysis buffer, then pooled for each biological replicate. RNA concentrations of pooled pellets were measured using the Qubit RNA Broad Range assay (ThermoFisher Scientific, #Q10210), then pellets were split back into equal volumes. For each biological replicate, equal amounts of RNA were digested either with Roche MNase (Nuclease S7, Roche, #10107921001) or with MNaseA, for 1 h at 25 °C, at a ratio of 250 U per 1 μg RNA. Digested pellets were loaded onto a 10-50% sucrose gradient (sucrose dissolved in 1× lysis buffer) and ultracentrifuged using a SW41 Ti rotor (Beckman Coulter) at 40,000 rpm (273,620 x g) for 2.5 h at 4°C. Gradients were fractionated into 450 μL fractions to resolve ribosome 30S and 50S subunits, monosomes, and polysomes. Fractions were flash-frozen on dry ice for subsequent RNA extraction. TRIzol LS (Invitrogen) was used according to manufacturer instructions to extract RNA from sucrose gradient fractions corresponding to 70S monosomes. Library preparation was performed as previously described (Gelsinger et al. 2022). Briefly, 1-5 µg of RNA fragments extracted from 70S monosome fractions were used to purify 10–45 nt RNA fragments by polyacrylamide gel electrophoresis on a 15% TBE-Urea gel; RNA fragments were treated with T4 polynucleotide kinase (NEB, #), ligated to the linker (NEB Universal miRNA Cloning Linker, #S13115S) using T4 RNA ligase (NEB, #M0242), purified by an Oligo Clean and Concentrator kit (Zymo Research, #D4061), and reverse transcribed with SuperScript III (Invitrogen, #18080044) using custom primers previously described (Gelsinger et al. 2022). DNA fragments were gel purified on a 10% TBE-Urea denaturing polyacrylamide gel, circularized using CircLigase (Epicentre, CL4115K), and PCR amplified (8–12 cycles) with Phusion polymerase (NEB) using custom primers (Gelsinger et al. 2022). PCR products were gel extracted from a native 8% TBE gel and analyzed for size and concentration using a BioAnalyzer high-sensitivity DNA kit (standard protocol) before sequencing (Novagene, Novaseq 6000, PE150).