Name: untreated.rep2.ribo.40s.libE_s
Instrument: Illumina NovaSeq 6000
Strategy: OTHER
Source: TRANSCRIPTOMIC
Selection: other
Layout: SINGLE
Construction protocol: Cells were treated with 3% PFA for 10 min, quenched with glycine and pelleted Cells were grown at 32C in EMM2 (Moreno et al. Methods Enzymology 194: 795). Between 3x10E8 and 12x10E8 cells were resuspended in 100 ul of lysis buffer (25 mM Hepes-KOH pH 7.6, 100 mM KCl, 2 mM MgCl2, 0.25% triton-100, 0.5 mM DTT, 250 mM glycine, Complete Mini EDTA Free Protease Inhibitor Cocktail (Roche), 10 mM PMSF, 100 µg/mL cycloheximide, 100 U Ribolock RNase inhibitor (ThermoFisher), and 1 U TURBO DNase (ThermoFisher)) with 1 g of chilled glass beads (Biospec) and lysed using a Fastprep 5G bead-beater (MP Biomedicals) at level 7 for 13 seconds. The extract was diluted with 200 ul of lysis buffer and cleared by centrifugation in two steps at 4 degrees C at 16,000 g (10 minutes followed by 15 minutes). 600 A260 units of cell extract were digested with 325 units of RNase I (Life Technologies) for 45 minutes. Reactions were quenched with 600 units of SUPERaseIn (Life Technologies). Digested extracts in 500 ul were loaded onto a 14 ml linear 7.5-30% (w/v) sucrose gradient prepared with a Gradient Master (Biocomp), and separated by centrifugation for 4 hr at 37,000 rpm in a SW 40Ti rotor (Beckman). The gradients were then fractionated by upward displacement with 55% (w/v) sucrose, and fractions containing 40S and 80S complexes were selected for further processing. RNAs were then purified by phenol extraction at 65C for 45 min, and run on 10% TBE-urea gels (Life Technologies). Fragments of 15-100 nucleotides were extracted from the gel Gel purified RNA fragments were treated with 10 units of T4 PNK (Thermo Fisher) in a low pH buffer (700 mM Tris pH 7, 50 mM DTT, 100 mM MgCl2) for 30 min at 37C. ATP and buffer A (Thermo Fisher) were then added for an additional 30 min incubation. RNA fragments were column-purified (PureLink RNA micro-columns, Life Technologies). 100 ng were used as input for the NEXTflex Small RNA Sequencing Kit v3 (Bioo Scientific), following manufacturer's no size selection protocol.