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Status |
Public on Aug 22, 2019 |
Title |
atp2 mutant TRiC total Rep 2 |
Sample type |
SRA |
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Source name |
atp2 mutant TRiC total
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Organism |
Saccharomyces cerevisiae |
Characteristics |
strain: BY4741 genotype/variation: atp2-P353A,P355A treatment: Cells were treated with 100 µg/mL cycloheximide (CHX) and immediately harvested using fast filtration. 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 |
SAMN09254303 |
SRA |
SRX4121350 |
Supplementary file |
Size |
Download |
File type/resource |
GSM3152898_atp2_R2.txt.gz |
12.3 Mb |
(ftp)(http) |
TXT |
SRA Run Selector |
Raw data are available in SRA |
Processed data provided as supplementary file |
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