|
| Status |
Public on Jun 10, 2014 |
| Title |
Anisomycin replicate 1B |
| Sample type |
SRA |
| |
|
| Source name |
Anisomycin
|
| Organism |
Saccharomyces cerevisiae |
| Characteristics |
strain: BY4741
treatment: anisomycin
|
| Treatment protocol |
Anisomycin was added to mid-log cells at 100 µg/mL and cells were allowed to grow for an additional 30 minutes prior to harvest. Anisomycin was also present at 100 µg/mL in the lysis buffer.
|
| 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 HiSeq 2000 |
| |
|
| Description |
anisomycin_1A and anisomycin_1B are technical replicates from the same culture.
|
| 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 |
| E-mail(s) |
lareau@berkeley.edu
|
| 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 |
GPL13821 |
| Series (1) |
| GSE58321 |
Distinct stages of the translation elongation cycle revealed by sequencing ribosome-protected mRNA fragments |
|
| Relations |
| BioSample |
SAMN02846431 |
| SRA |
SRX581796 |
