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Sample GSM2895491

Query DataSets for GSM2895491

Status

Public on Dec 21, 2017

Title

ribo_37_WT_w/o_CHX_2

Sample type

SRA

Source name

ribo_37_WT_w/o_CHX_2

Organism

Saccharomyces cerevisiae

Characteristics

strain: BY4741

Treatment protocol

No additional treatment

Growth protocol

Yeast cells were generally grown to an OD600 of ~ 0.7 in synthetic complete medium at 37°C,without treatment with cycloheximide before harvesting.

Extracted molecule

total RNA

Extraction protocol

Cells were rapidly vacuum filtered and flash frozen. Whole cell extracts were prepared by breaking cells in freezer mill in cell lysis buffer containing 500 µg/ml cycloheximide. Whole cell extracts were RNase treated, and run over sucrose gradients to extract the monosome peak. RNA was then extracted using SDS/hot phenol/chloroform and footprints of the specified size were retained by gel purification. Briefly, RNAs were ligated to a universal DNA linker followed by reverse transcription. cDNAs were then circularized and PCR amplified. Ribosome profiling circularized cDNA was subject to ribosomal RNA subtraction using custom biotinylated oligos.

Library strategy

OTHER

Library source

transcriptomic

Library selection

other

Instrument model

Illumina HiSeq 2500

Data processing

The ribosome profiling and mRNA sequencing data were processed as described in Ingolia, N.T. (2010). Genome-wide translational profiling by ribosome footprinting. Methods Enzymol. 470, 119–142. Breifly, the sequnce linker and the first nucleotide of each read were removed using fastx_toolkit/0.0.13 (fastx_clipper and fastx_trimmer, respectively). Ribosome RNAs were removed using bowtie/2-2.2.5 alignment agains year ribosome RNA sequences.The non-rRNA reads were mapped to yeast geneome sacCer3 (http://hgdownload.soe.ucsc.edu/goldenPath/sacCer3/bigZips/chromFa.tar.gz)using TopHat/2.0.13.
Wiggle files were produced from the alignment files using RiboSeq (https://github.com/ingolia-lab/RiboSeq), Two replicates of mRNA or foot-print alignment files were merged for generating two wiggle files, each for genes on the Watson or Crick strand. The total reads on both strands were summed and a normalization factor q was calculated as 1000,000,000/(total reads on W+C strands). Wiggle files were then regenerated by multiplying all reads by the factor q, yielding the number of reads per 1000 million total reads (rpkm).
For uORF identification, we employed the "yassour-uorf" program in RiboSeq (https://github.com/ingolia-lab/RiboSeq), to identify all potential uORFs within annotated 5’UTRs initiating with either AUG or a near-cognate codons. We use the perameter -c15-r4-z0.5 in the relevant line of code. This analysis was conducted on multiple published datasets listed in (Dataset-list.docx) and all unpublished ribosome profiling datasets in this study. After excluding uORFs shorter than 3 codons, we identified 564 AUG initiated uORFs and 5497 near-cognate uORFs with evidence of translation in one or more experiments. The characteristics of all uORFs, including uORF_name, gene_name, position from 5' end (cap), position to main AUG, uORF_length, context, start codon and start_codon_type are listed in supplimentary file "all_uorf_info.xlsx".
Genome_build: sacCer3
Supplementary_files_format_and_content: a bed file for all yeast uORFs
Supplementary_files_format_and_content: wig
Supplementary_files_format_and_content: "Dataset-list.pdf" is for the published datasets we used in uORF identification, and “all_uorf_info.xlsx” listed all information for the uORFs we identified

Submission date

Dec 20, 2017

Last update date

Dec 22, 2017

Contact name

Jon R. Lorsch

E-mail(s)

jon.lorsch@nih.gov

Phone

301-594-2172

Organization name

National Institutes of Health