High throughput sequenceing of fission yeast to survey the dynamic repertoire of a eukaryotic transcriptome at the single nucleotide resolution
High throughput sequenceing of fission yeast to survey the dynamic repertoire of a eukaryotic transcriptome at the single nucleotide resolution
All cDNA samples used for Illumina sequencing were prepared by first treating ~1 mg of total RNA for 30 min with amplification grade RNase-free DNase (Invitrogen), according to the manufacturer’s protocols. PolyA enriched RNA was then prepared using an oligo(dT) selection kit (Oligotex Direct mRNA MiniKit, Qiagen). The resulting poly(A)-enriched RNA was then converted to double-stranded cDNA using a cDNA synthesis kit (Superscript choice system for cDNA synthesis, Invitrogen) , primed by an oligo(dT) primer, according to the manufacturer’s protocols. Random-primed RNA from rapidly proliferating cells was treated with an enzyme that specifically degrades non-capped RNA molecules (mRNA-only™ Eukaryotic mRNA isolation kit, Epicentre Biotechnologies) according to the manufacturer’s protocols. The resulting cDNA was then converted to double-stranded cDNA as above, except that the reaction was primed with random hexamers. RNA samples from the pooled meiotic timepoints were subjected to IVT amplification after undergoing a poly(A) enrichment step as described above. RNA from the oxidative stress condition and prp2 mutant was treated in the same way as the rapidly proliferating samples with oligo(dT)-primed cDNA synthesis after mRNA enrichment.
Experimental Factor: TIME
0 h
Experimental Factor: GROWTHCONDITION
normal
Experimental Factor: TEMPERATURE
32 degree_C
Experimental Factor: MEDIA
YE
Experimental Factor: CULTURE_DENSITY
0.2 OD