Total cellular RNA was extracted using hot acid-phenol extraction and the resulting RNA was chemically fragmented. Samples were barcoded using a 3’ adaptor with a unique sequence corresponding to each sample, and then pooled for downstream processing, as described in Shishkin et al., 2015 (link). rRNA was depleted from the pooled samples using the Illumina Ribo-Zero Gold rRNA Removal Kit for Yeast (MRZY1306). Sequencing was performed at the Functional Genomics Core at the University of Chicago. Detailed protocol for library preparation is available; see Shishkin et al., 2015 (link).
Ribo zero gold rrna removal kit for yeast
Manufactured by Illumina
The Ribo-Zero Gold rRNA Removal Kit for Yeast is a laboratory tool designed to selectively remove ribosomal RNA (rRNA) from yeast RNA samples. This kit is used to enrich for non-ribosomal RNA transcripts, enabling more efficient analysis of the yeast transcriptome.
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5 protocols using ribo zero gold rrna removal kit for yeast
1
RNA Fragmentation and Depletion Protocol
2
Ribosome Profiling and Transcriptome Analysis of Saccharomyces Species
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Ribosome profiling and mRNA sequencing experiments were conducted with S. cerevisiae strain YPS128 (Sniegowski et al. 2002 (link)) and with S. paradoxus strains YPS744, MSH604, and MSH587-1 belonging, respectively, to groups SpA, SpB, and SpC according to Leducq et al. (2016) (link). We prepared two replicates per strain and library type (see Methods) (Supplemental Methods ). Strains were grown in SOE medium (Murphy et al. 2006 (link)). Ribosome profiling footprints were purified using the protocol described by Baudin-Baillieu et al. (2016 (link); see Supplemental Methods ). The rRNAs were depleted in purified ribosome footprints and total mRNA samples using the Ribo-Zero Gold rRNA Removal Kit for yeast (Illumina). Ribosome profiling and total mRNA libraries were constructed using the TruSeq Ribo Profile kit for yeast (Illumina), using the manufacturer's instructions starting from the fragmentation and end repair step. Libraries were sequenced on an Illumina HiSeq 2500 platform at the Génome Québec Innovation Centre (Montréal, Canada). Raw reads are available from the NCBI SRA database (https://www.ncbi.nlm.nih.gov/sra ) under accession numbers SRR5996796 to SRR5996803 for ribosome profiling and SRR6022778 to SRR6022785 for total mRNA experiments.
3
RNA-seq of Yeast Transcriptome
4
Polyadenylation and rRNA Depletion of RNA
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5 μl of TURBO DNase treated RNA was polyadenylated with E. coli PAP (E-PAP, ThermoFisher Scientific) in 20 μl reactions, containing 1x reaction buffer, 2.5 mM MnCl2; 0.4 U E-PAP and 0.8 U RiboLock RNase inhibitor (ThermoFisher Scientific) for 30 min at 30°C. Reactions were then purified using a PureLink micro RNA purification kit (Ambion) and eluted using 22 μl RNase-free water. PureLink eluates were rRNA depleted using a down-scaled reaction of the Ribo-Zero Gold rRNA Removal Kit for Yeast (Illumina). That is, 18 μl of RNA was incubated with 2 μl of reaction buffer, 2 μl of removal solution and 8 μl of water for 10 min at 65°C, and put to RT for 5 min. This reaction was cleared using 65 μl of rRNA magnetic beads for 5 min at RT and 5 min at 50°C. Final samples were precipitated using NaOAc and ethanol with 20 μg of glycogen as carrier and resuspended in 10 μl of RNA storage solution.
5
Purification and Sequencing of 4tU-labeled RNA
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RNA quality of total and purified 4tU labeled RNA was tested and quantified using a Bioanalyzer (Agilent). Depending on the sample 200-350 ng of RNA was used to prepare libraries. To generate pA- data, an aliquot of the 2 min 4tU labeled RNAs was in vitro polyadenylated using Escherichia coli poly(A) polymerase (AM1350; ThermoFisher) according to the manufacturer’s instructions. Reactions were then purified using PureLink micro RNA purification kit from Ambion (12183018A) and ribo-depleted using Ribo-Zero Gold rRNA Removal kit for yeast (MRZY1306) from Ilumina according to the manufacturer’s instructions. All libraries were then prepared using Lexogen QuantSeq 3′ mRNA-Seq Library Prep Kit REV (016.96, Lexogen GmbH) for Illumina and sequenced at Vienna Biocenter Core Facilities (Vienna, Austria) multiplexing 32 samples on a HiSeqV4 SR50 (single end 50nt reads) run and using the QuantSeq REV specific primer CSP. An overview of samples, read quality control and mapping statistics are provided in Table S1 and Table S2 .
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