Illumina Genome Analyzer II sequencing; Genome-wide Analysis of Core Promoter Structures in Schizosaccharomyces pombe with DeepCAGE
Genome-wide Analysis of Core Promoter Structures in Schizosaccharomyces pombe with DeepCAGE
We used a wild-type Schizosaccharomyces pombe 972 h- strain for all experiments. The strain was exponentially grown at 32 degree C in rich medium supplemented with glucose, adenine, histidine, lecucine and uracil. Cells were harvested and resuspended with pre-cooled lysis buffer (10mM EDTA, 10 mM Tris-HCl pH 8.0 and 50 mM NaOAc), which were further treated with mechanical pulverization.  The cell material was stored at -80 degree C. Total RNA was extracted with the TRIZOL reagent according to the manufacturer's instructions (Invitrogen, Carlsbad, CA) and isolated RNA was quantified by bioanalyzer 2100. The CAGE library was prepared as the method described (Valen et al., Genome Res 2009) with some modifications to work with Illumina GA II sequencer. Briefly, cDNA synthesis was conducted by using 50ug total RNA and Superscript II revese transcriptase (Life Technologies). The cap selected cDNA/RNA hybrids were treated with RNase ONE Ribonuclease (promega) and purified cDNA was ligated with 5 prime end linker containing Mme I site. After 2nd strand synthesis, dsDNA fragments were digested with Mme I followed by the ligation with 3 prime end linker. The resulting CAGE tags were amplified by PCR, which was subject to deep sequencing.
Experimental Factor: growth condition
Normal