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| Status |
Public on Aug 22, 2019 |
| Title |
Ssb-bound puromycin Rep 1 |
| Sample type |
SRA |
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| Source name |
Ssb-bound puromycin
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| Organism |
Saccharomyces cerevisiae |
| Characteristics |
strain: BY4741
genotype/variation: wild-type
treatment: Lysis buffer supplemented with 5 mM puromycin.
molecule subtype: Ribosome protected mRNA
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| Growth protocol |
Yeast were grown at 30°C in YPD until mid-log phase, followed by harvesting using fast filtration and flash freezing in liquid nitrogen.
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| Extracted molecule |
total RNA |
| Extraction protocol |
Following cell lysis, samples were digested with RNase I and ribosomal pellets were isolated by ultracentrifugation using a sucrose cushion (or sucrose gradient for WT translatome samples). As indicated, TRiC- or Ssb-bound ribosomes were immunoprecipitated, followed by RNA extraction.
RNA fragments in the range of 24-35 nucleotides were gel purified. After dephosphorylation, a DNA linker (CTGTAGGCACCATCAAT) was ligated to the 3' end of the RNA footprints, which were then reverse transcribed into cDNA, and circularized. Depletion of rRNA was performed using the Ribo-Zero Gold kit (Illumina) before linker ligation, or using biotinylated subtraction oligonucleotides after circularization for WT translatome libraries. Libraries were then barcoded during PCR amplification and sent for sequencing.
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| Library strategy |
OTHER |
| Library source |
transcriptomic |
| Library selection |
other |
| Instrument model |
Illumina HiSeq 4000 |
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| Data processing |
Illumina CASAVA 1.8 was used for base calling and demultiplexing.
Adaptor sequences (CTGTAGGCACCATCAAT) were trimmed using Cutadapt v1.4.2, followed by removal of the 5' nucleotide using FASTX-Trimmer.
Ribosomal RNA reads were removed by alignment using Bowtie v1.0.0.
Remaining reads were aligned using Bowtie v1.0.0 (parameters "-y -a -m 1 -v 2 --norc --best --strata") to a library of open reading frames that excluded dubious ORFs and ORFs that overlapped with other genes.
A nucleotide offset was empirically calculated from the 5' end of each fragment length that displayed three nucleotide periodicity, thereby assigning each read to a specific A-site nucleotide. Nucleotide reads were then summed for each codon.
Genome_build: sacCer3 R64-1-1
Supplementary_files_format_and_content: Tab-delimited text files include: Column1: ORF; Column2: codon position of each ORF, including 21 flanking nucleotides (7 triplets) before the start codon and after the stop codon; Column3: codon; Column4: counts of ribosome reads.
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| Submission date |
May 24, 2018 |
| Last update date |
Aug 24, 2019 |
| Contact name |
Kevin C. Stein |
| E-mail(s) |
kcstein@stanford.edu
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| Organization name |
Stanford University
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| Department |
Biology
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| Lab |
Frydman
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| Street address |
Clark Center E200, 318 Campus Drive
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| City |
Stanford |
| State/province |
California |
| ZIP/Postal code |
94305 |
| Country |
USA |
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| Platform ID |
GPL21656 |
| Series (1) |
| GSE114882 |
Nascent Polypeptide Domain Topology and Elongation Rate Direct the Cotranslational Hierarchy of Hsp70 and TRiC/CCT |
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| Relations |
| BioSample |
SAMN09254314 |
| SRA |
SRX4121343 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM3152891_Ssb_S1puro.txt.gz |
10.7 Mb |
(ftp)(http) |
TXT |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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