NextSeq 550 sequencing; Ribosome profiling and parallel RNA-seq of S. pombe cells subject to amino acid starvation
Ribosome profiling and parallel RNA-seq of S. pombe cells subject to amino acid starvation
Cells were incubated with CHX at 100 ug/ul for 5 minutes before collection 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 (20 mM Tris-HCl pH 8.0, 140 mM KCl, 5 mM Mg2Cl, 1 % Triton X-100, 100 ug/ml cycloheximide) with 1 g of chilled glass beads (Biospec) and lysed using a Fastprep 24 bead-beater (MP Biomedicals) at level 6 for 13 seconds. The extract was diluted with 400 ul of lysis buffer and cleared by centrifugation in two steps at 4 degrees C at 16,000 g (5 minutes followed by 15 minutes). 600 A260 units of cell extract were digested with 750 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 10-50% (w/v) sucrose gradient prepared with a Gradient Master (Biocomp), and separated by centrifugation for 160 min at 35,000 rpm in a SW 40Ti rotor (Beckman). The gradients were then fractionated by upward displacement with 55% (w/v) sucrose, and fractions containing monosomes selected for further processing. RNAs were then purified by phenol extraction, passed through amicon ultra 100 kDa columns (Millipore), and run on 15% TBE-urea gels (Life Technologies). Fragments of around 28 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 v2 (Bioo Scientific), following manufacturer's protocol.