title: Illumina Genome Analyzer II sequencing; Pombe_ribosome_profiling_vegetative_cells
design: Pombe_ribosome_profiling_vegetative_cells
library_construction_protocol: Cells were grown in yeast extract medium (Moreno et al., 1991, Methods Enzymol. 194, 795-823) at 32C,  incubated for 5 minutes with 100 ug/ml cycloheximide, pelleted at 4C and frozen in liquid N2 3x10E8 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) 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 4C at 16,000 g (5 minutes followed by 15 minutes).  600 A260 units of wild type vegetative cell extract were digested with 750 units of RNase I (Life Technologies) for 10 minutes. Reactions were quenched with 600 units of SUPERaseIn (Life Technologies).  Digested extracts in 500 ?l 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 a YM-100 column (Millipore), and run on 15% TBE-urea gels (Life Technologies).  Fragments of around 28 nucleotides were extracted from the gel. Both mRNA and ribosomal footprint gel-purified samples were processed identically.  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.
attributes.tag: Experimental Factor: RNA
attributes.value: ribosome-protected RNA