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Sample GSM1406455 Query DataSets for GSM1406455
Status Public on Jun 10, 2014
Title Untreated replicate 3
Sample type SRA
Source name Untreated
Organism Saccharomyces cerevisiae
Characteristics strain: BY4741
treatment: untreated
Treatment protocol untreated
Growth protocol Yeast were innoculated from an overnight culture into 500mL of YEPD media then grown at 30C from a starting OD of 0.2 to a final OD of ~0.6.
Extracted molecule total RNA
Extraction protocol Cells were harvested by filtration then frozen in liquid N2 and pulverized using a Retsch mixer mill. Lysis buffer was added to cell powder and lysates were clarified by centrifugation. RNAse I was added to cell lysate. After RNAse digestion, lysate was sedimented in a linear 10%-50% sucrose gradient at 35000rpm for 3 hours. Gradients were fractionated and the monosome peak fraction was collected. RNA was purified using a miRNeasy Mini kit from Qiagen.
RNA was size selected using 18 and 34 nt size markers then treated to remove the 3’ phosphate and ligated to the Universal miRNA cloning linker from New England Biolabs. Ligated fragments were reverse transcribed using a primer complementary to the cloning linker which introduced sequencing adapters. RNA template was removed and cDNA was circularized using CircLigase ssDNA ligase from Epicentre. Ribosomal RNAs were removed by hybridization to subtraction oligos. Libraries were amplified with PCR. Libraries were then sequenced on an Illumina Genome Analyzer 2 or an Illumina HiSeq 2000.
Library strategy RNA-Seq
Library source transcriptomic
Library selection cDNA
Instrument model Illumina Genome Analyzer II
Data processing library strategy: Ribo-Seq (ribosome profiling)
Cloning linker sequences were trimmed from Illumina reads. Trimmed sequences were aligned to S. cerevisiae ribosomal and noncoding RNA sequences using bowtie v. 0.12.7 or v. 1.0.0 to remove rRNA reads. The non-rRNA reads were aligned to the S. cerevisiae genome as a first pass to remove any reads that mapped to multiple locations. Reads that passed this filter were then aligned to the S. cerevisiae transcriptome with bowtie, allowing two mismatches and only reporting alignments of reads that mapped uniquely in the transcriptome (bowtie -v 2 -m 1 -a --norc --best –strata). A list of read counts and read lengths per nucleotide position in the transcriptome, based on the 5’ end of the mapped read, was generated.
The counts of small and large footprints from ribosomes with each codon positioned in the inferred A site were generated from the list of reads at each nucleotide position. The large footprints were defined as 28 nt reads with the 5’ end 15 nt upstream of the codon at position i, and 29 nt reads with the 5’ end 16 nt upstream of i. Small footprints included 20 nt and 21 nt reads with the 5’ end 15 nt upstream of i and 21 nt and 22 nt reads with the 5’ end 16 nt upstream of i. For each gene, the analysis included codons 51 through the second codon before the stop codon, to avoid the region at the beginning of genes from which ribosomes have been depleted by runoff elongation during harvest. Genes with fewer than 10 footprints in total were excluded, as were any non-unique 20mer regions within genes.
Genome_build: sacCer2
Supplementary_files_format_and_content: A percodon.txt file for each sample provides the count of small and large ribosome footprints at each codon, starting at the 51st codon of each gene. Files include gene symbol, codon position, codon sequence, and count of small and large ribosome footprints with that position in the inferred A site. Genes with fewer than 10 footprints are excluded.
Submission date Jun 09, 2014
Last update date May 15, 2019
Contact name Liana F Lareau
Organization name University of California, Berkeley
Department California Institute for Quantitative Biosciences
Street address Barker Hall 424
City Berkeley
State/province CA
ZIP/Postal code 94720
Country USA
Platform ID GPL9377
Series (1)
GSE58321 Distinct stages of the translation elongation cycle revealed by sequencing ribosome-protected mRNA fragments
BioSample SAMN02846425
SRA SRX581792

Supplementary file Size Download File type/resource
GSM1406455_untreated_3.percodon.txt.gz 5.0 Mb (ftp)(http) TXT
SRA Run SelectorHelp
Raw data are available in SRA
Processed data provided as supplementary file

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