GSM2544923: S. pombe G34R cells_BRL107; Schizosaccharomyces pombe; RNA-Seq
The cells were pelleted by centrifugation and washed in DEPC H2O. The pellet was resuspended in 750ul TES Buffer [50mM Tris-HCl pH 7.5, 10mM EDTA, 100mM NaCl, 0.5% SDS made in DEPC H2O] along with an equal volume of 5:1 phenol:chloroform pH 4.7 and incubated at 65°C for one hour with vortexing for 10 seconds every 10 minutes. The samples were then cooled on ice and centrifuged for five minutes at 13,000 RPM. The aqueous phase was transferred to a 2mL phase lock tube and an additional phenol:chloroform extraction was performed. After centrifugation, the aqueous phase was transferred to a new tube and an equal volume of chloroform was added. To precipitate the RNA, the aqueous phase was transferred to a 2mL microcentrifuge tube and three volumes of ice cold ethanol and 3M NaOAc pH 5.2 were added and the samples were kept at -20°C to precipitate the RNA. The next day, samples were centrifuged at 14,000 RPM and 4°C for 15 minutes to pellet the RNA. The pellet was washed with ice cold 70% ethanol and air dried for 30 minutes. A Turbo DNase (Ambion) reaction was set up with 100ug of RNA in a 150ul reaction containing 5ul of Turbo DNase. The reaction was incubated at 37°C for 30 minutes and another 5ul of Turbo DNase was added and incubated for an additional 30 minutes prior to the removal of the DNase using 50ul of inactivation beads. An RNeasy Mini kit (Sigma) was used to further clean up and concentrate the RNA which was eluted in a final volume of 30ul of DEPC H2O. Libraries were prepared from 2ug of RNA. Ribosomal RNA was removed from the samples using Ribo-Zero Gold rRNA Removal Kit (Yeast) following manufacturer instructions (Illumina). Libraries were prepared using the TruSeq Strand Total RNA Library Prep Kit, beginning at Elution 2 – Fragment – Prime step immediately preceding cDNA synthesis according to the manufacturer instructions (Illumina) with the following modifications; the 94 C Elution 2 – Fragment – Prime incubation was reduced to five minutes and the PCR was reduced to 11 cycles.