Illumina Genome Analyzer IIx sequencing; Total and 4-thiouracil (4tU) labeled RNA samples of wild-type Schizosaccharomyces pombe strain ED666 (BIONEER)
Total and 4-thiouracil (4tU) labeled RNA samples of wild-type Schizosaccharomyces pombe strain ED666 (BIONEER)
A fresh plate (YES) was inoculated from glycerol stock. An over-night culture was inoculated (YES medium) from a single colony and grown at 30 ???C.  In the morning a 120 mL culture (YEA medium) was started at OD600 0.1 and grown to OD600 of 0.8 at 32 ???C in a water bath at 150 rpm.  4-thiouracil was added to 110 mL of culture at 5 mM final concentration.  20 mL samples were taken out after 2, 4, 6, 8, and 10 minutes. Each sample was centrifuged immediately at 32 ???C, at 3,500 rpm for 1 min.  The supernatant was discarded and the pellet was frozen in liquid nitrogen. All experiments were performed in two independent biological replicates. Total RNA was extracted and samples were DNase digested with Turbo DNase (Ambion).  Labeled RNA was purified as published (Sun et al., 2012). rRNA was depleted using the Ribo-ZeroTM Gold Kit (Yeast, Epicentre) according to  the manufacturer???s recommendation with 1.5 ??g labeled RNA and with 2.5 ??g total RNA as input.  Sequencing libraries for the time series samples were prepared according to the manufacturer???s recommendations using the ScriptSeq??? v2 RNA-Seq Library Preparation Kit (Epicentre).
Experimental Factor: 4tU
labelling
Experimental Factor: 2
time