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. RNA samples were purified using Purelink RNA microcolumns (Life Technologies) as described by the manufacturer, except that the samples were initially passed through the column in the presence of 70% ethanol (to favour binding of small RNAs).  Samples were then treated with poly nucleotide kinase (PNK, Fermentas) and polyadenylated using 12 units of poly-(A) polymerase (NEB) at 37C for 45 minutes.  Reverse transcription reactions were performed using custom primers containing an anchored oligo(dT), 4 nucleotides of known sequence used for multiplexing, and 5 random nucleotides that serve as unique molecular identifiers.  Reverse transcription products were gel-purified and circularised using CircLigase II (Epicentre), and amplified by PCR with custom library primers for 12 or 15 cycles