GSM2177925: 6C_TP; Saccharomyces cerevisiae; RNA-Seq
For the mRNA libraries, cells were lysed and total RNA isolated using the RNeasy kit (Qiagen, Cat#: 74104), which was then used to isolate poly(A)+ RNA with the Oligotex kit (Qiagen, Cat#: 70022). The purified mRNA from each sample was then used with the ScriptSeq v2 RNA-Seq library preparation kit (Illumina, Cat#: SSV21124), exactly according to the manufacturer’s instructions. In the final PCR amplification of the libraries, the forward primer was the one included in the ScriptSeq v2 RNA-Seq kit, while the reverse index PCR primers used for the mRNA libraries were primers 1 through 8, from the ScriptSeq Set 1 (Illumina, Cat#: RSBC10948) For the ribosome footprint libraries, the cell extracts were prepared as follows: The cell pellets were in 1.5 ml screw-cap microcentrifuge tubes and they were washed once with 1 ml ice-cold DEPC-treated water, centrifuged at 13,000 g for 1 min and the supernatant was aspirated. To each tube we added 0.25 ml glass beads (425-600 μm; Sigma-Aldrich, Cat#: G9268) and 0.4 ml ice-cold lysis buffer, which is described in Ingolia et al(Ingolia et al., 2012). The cells were broken by vortexing at full speed eight times, for 15 sec each time. The samples were placed in ice for at least 30 sec between each vortexing. The extracts were clarified by centrifugation at 5,000 g for 5 min, at 4 °C. The supernatant was clarified in a second centrifugation, at 13,000 g for 10 min, at 4 °C. All subsequent steps for the ribosome footprint libraries were performed as described in Ingolia et al(Ingolia et al., 2012), except step 7, where we used 1.875 μl of RNase I (100 U μl-1) per reaction. For all gel extractions, we used the overnight protocol described in(Ingolia et al., 2012). Depletion of rRNA was done as described in (Ingolia et al., 2012). In the final PCR amplification and barcode addition step, we amplified each library for 12 cycles
GEO Accession
GSM2177925