GSM8880366: H98 ribosome footprints, 2mM H2O2, biol rep 2; Haloferax volcanii; OTHER
Cultures were flash frozen in liquid nitrogen and lysed by cryogenic mill, using 55 g of frozen culture and 100 µg/mL of anisomycin. Ribosomes were collected by ultracentrifugation on a 1 M sucrose cushion. Polysomes were digested using MNase and separated using a 10-50% sucrose gradient. RNA from monosome fractions was isolated with Trizol LS reagent. Size selection (15-45 nt) of RNA extracted from monosome fractions was performed by polyacrylamide gel electrophoresis, followed by rRNA reduction using the Ribo-zero (Bacteria) kit (Illumina, discontinued). RNA fragments were ligated to a univeral cloning linker (NEB Universal miRNA Cloning Linker, #S13115S), and reverse transcribed using SuperScript III (Invitrogen, #18080044). DNA was then circularized using CircLigase (Epicentre, CL4115K), and PCR amplified.