GSM8461881: biofilm-rna-3; Candida albicans; RNA-Seq
Cell lysates were prepared as described previously ((Mundodi, et al., G3 11(2):jkaa043 (2021) and Choudhary, et al., Microbiol. Spectr. 11(5):e0257223 (2023)), except that samples were lysed by bead beating 6 times for 1 min. each with a 1 min. rest on ice between vortexes. Cells were recovered by filtration at room temperature, rapidly scraped off the filter paper with a cell scraper, placed into ice-cold lysis buffer (1x yeast polysome buffer (Illumina), 1% Triton X-100, (Sigma), 50 ug/mL GMPPNP (Sigma), 10 ug/mL Blasticidin S (InvivoGen)), and  homogeneously mixed, followed by snap freezing in liquid nitrogen. Cell lysates were prepared and ribosome profiling was performed using three biological replicates as we have described previously (Mundodi, et al., G3 11(2):jkaa043 (2021) and Choudhary, et al., Microbiol. Spectr. 11(5):e0257223 (2023)). 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 (see Dataset S8 in Mundodi, et al., G3 11(2):jkaa043 (2021)). 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.