s52a.control.ribo.2
Schizosaccharomyces pombe
Protocols: Cells were collected by filtration. Filters were immediately frozen in liquid nitrogen. Cells were grown at 32C in YE5S (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.
ENA-FIRST-PUBLIC
2020-06-01T04:03:53Z
ENA-LAST-UPDATE
2020-01-28T15:59:41Z
External Id
SAMEA6506057
INSDC center name
Department of Biochemistry University of Cambridge UK
INSDC first public
2020-06-01T04:03:53Z
INSDC last update
2020-01-28T15:59:41Z
INSDC status
public
Submitter Id
E-MTAB-8745:s52a.control.ribo.2
broker name
ArrayExpress
common name
fission yeast
fraction
ribosome protected fragments
genotype
eIF2alpha-S52A
replicate
2
sample name
E-MTAB-8745:s52a.control.ribo.2
scientific_name
Schizosaccharomyces pombe