Schizosaccharomyces pombe
Protocols: Cells were grown in Edinburgh Minimal Medium (EMM; Moreno et al., 1991, Methods Enzymol. 194, 795-823.) at 32C. RNA was extracted using a hot phenol extraction method as described in Lyne et al. (2003) BMC Genomics 4:27 Total RNA was prepared using a hot-phenol extraction protocol. 10 ug of poly(A) selected mRNA were incubated with 1/9 volume of RNA fragmentation reagents (Ambion, Life Technologies) at 70 degrees C for 4 min 30 sec. The reaction was stopped by adding 1/9 volume of stop solution (Ambion, Life Technologies) and transferring the samples to 4 degrees C. Polyadenylated fragmented RNA was purified using oligo(dT) magnetic beads (Life Technologies). 200 ul of beads were washed in binding buffer (20 mM Tris-HCl pH 7.0, 2 mM EDTA, 1 M LiCl). 50 ul of binding buffer was added to 50 ul of fragmented RNA, and the mix was incubated at 65 degrees C for 2 minutes and then transferred on ice. The RNA was then added to the beads, and incubated at room temperature for 5 minutes. The beads were washed twice in wash buffer (10 mM Tris-HCl pH 7.0, 1 mM EDTA, 0.15 M LiCl). RNA was eluted by resuspending the beads in 22 ul of 10 mM Tris pH 8.0 and incubating at 80 degrees C for 2 minutes. The remaining RNA in lysis buffer was incubated with the beads once more and washed as described above, and the two eluates combined. For reverse transcription, 11 ul of purified RNA fragments were mixed with 1 ul of 10 mM dNTPs and 1 ul of 50 uM RT primer, incubated at 65 degrees C for 5 minutes and transferred to ice. 4 ul of 5X FFS buffer (Invitrogen, Life Technologies), 1 ul of SuperaseIN (Ambion, Life Technologies), 1 ul of 0.1 M DTT and 1 ul of Superscript III (Invitrogen, Life Technologies) were added, and the reaction incubated at 48 degrees C for 40 minutes. RNA was hydrolyzed by adding 2.3 ul of 1 M NaOH and incubating at 95 degrees C for 15 minutes, and the solution neutralized with 2.3 ul of 1 M HCl. The cDNA was purified using Agencourt AMPure magnetic beads (Beckman Coulter). 46 ul of beads were added to the cDNA solution (~1.85 volumes), and the beads washed twice with 180 ul of freshly prepared 70 % ethanol. The cDNA was eluted by resuspending the beads in 40 ul of water. A second round of purification was performed as above, except that a lower ratio of beads to sample (1.6 volumes) was used. This second round is essential for the complete removal of leftover RT primer, which can otherwise circularise and be amplified by PCR. 8 ul of cDNA were circularised using Circligase II (Epicentre) as follows: 0.5 ul of 50 mM MnCl2, 1 ul of 10X Reaction buffer and 0.5 ul of enzyme were added, and the reaction incubated at 60 degrees C for 60 minutes and at 80 degrees C for 10 minutes. PCR amplification was performed using Phusion High Fidelity Polymerase (Thermo Scientific). 2 ul of circularised cDNA were used as a template, and amplified in a 50 ul volume as described by the manufacture. Primers P3 and P5 were used at a final concentration of 0.2 uM. PCR amplification was performed in two steps: four cycles using an annealing temperature of 66 degrees C, followed by a variable number of cycles at 72 degrees C. Small scale reactions were performed to determine the optimal number of cycles, which ranged between 12 and 18 (including both stages).
ENA-FIRST-PUBLIC
2013-11-19T17:02:04Z
ENA-LAST-UPDATE
2018-03-08T16:23:19Z
External Id
SAMEA2065712
INSDC center name
Department of Biochemistry, University of Cambridge, UK
INSDC first public
2013-11-19T17:02:04Z
INSDC last update
2018-03-08T16:23:19Z
INSDC status
public
Submitter Id
E-MTAB-1642:wt_3
broker name
ArrayExpress
common name
fission yeast
sample name
E-MTAB-1642:wt_3
scientific_name
Schizosaccharomyces pombe
strain
972 h-