GSM4006538: 05C_TP_RPL22B; Saccharomyces cerevisiae; RNA-Seq
We used the same approach we had described previously (Blank et al., 2017), to collect cells from elutriated cultures of each ribosomal protein deletion and generate ribosome footprint libraries. For the RNAseq libraries from the same samples, we also used the same approach we had described (Blank et al., 2017), except that we did not select for polyA-tailed RNAs. Instead, from total RNA, we depleted rRNA, using the 'Ribo-Zero™ Magnetic Gold Kit (Yeast)', according to the manufacturer's instructions. All RNA-seq libraries were sequenced on an Illumina HiSeq4000, with multiplexing, at the Texas A&M AgriLife Genomics and Bioinformatics Facility 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
GSM4006538