title: GSM4672121: ribo_30C_2; Candida albicans; OTHER
library_construction_protocol: Lysates were prepared as described previously (Spealman et al. 2016) with a few modifications.  Flash frozen cells were thawed on an ice water slurry and cell suspensions were transferred into tubes with 0.5 mm diameter acid-washed glass beads and placed on ice for 5 min. The samples were lysed by vortexing 8 times for 30 sec. each with a 30 sec. rest on ice between each vortex. Samples were further processed according to instructions for the Illumina TrueSeq Ribo Profile (Yeast) Kit. Briefly, lysates were pre-cleared by centrifugation at 3000g for 5 min. at 4°C to remove cell debris and further clarified by centrifugation at 20000g for 10 min. at 4°C. The lysates were treated for 10 min. on ice with 10U/mL DNase I (Illumina) and quantified by measuring absorbance at 260 nm (A260). Finally, 100 uL aliquots were frozen in liquid nitrogen and stored at -80°C until further use. Ribosome profiling and library preparation were carried out according to the protocol described for the True Seq Ribo Profile (Yeast) Kit (Illumina). Lysates were digested with 15 U RNase I (Ambion) per A260 unit and incubated at room temperature for 45 min. with gentle rotation. The reactions were stopped by the addition of 1000U/mL SUPERase-IN (Ambion). 80S monosome fractions were purified from the cell lysate using MicroSpin S-400 HR columns (GE Healthcare). Ribosome protected fragments (RPFs) were further purified using the RNA Clean & Concentrator-25 Kit (Zymo Research) method. This kit was also used for total RNA purification. rRNA depletion was carried out using a Ribo-Zero Magnetic Gold (Yeast) kit (Illumina). An additional subtraction step was included to remove rRNA sequences from circularized cDNA as described previously (Spealman et al. 2016) with slight modifications.  Subtractions were carried out in a 30 uL reaction volume, with 10 uL sample, 2 uL 20X SSC and 2 uL of a rRNA subtraction pool containing custom-designed biotinylated oligonucleotides (Dataset S8). The circularized DNA was next used as a template for amplification of library PCR products. PCR products of 140-160 bp were recovered from excised gel slices, quantified by Agilent  Bioanalyzer/Fragment analyzer and deep sequencing was performed using an Illumina Hiseq3000 machine at the Greehey Children's Cancer Research Institute Genome Sequencing Facility (University of Texas Health Science Center at San Antonio). All ribosome profiling experiments were performed in biological triplicate.
identifiers.namespace: GEO
identifiers.id: GSM4672121