|
| Status |
Public on Dec 14, 2021 |
| Title |
cyt-1: cyt DHFR - with CHX - Replicate 1 |
| Sample type |
SRA |
| |
|
| Source name |
ribosome preparation
|
| Organism |
Saccharomyces cerevisiae |
| Characteristics |
replicate: 1
clogger expression: cytosolic DHFR (control)
chx treatment: 2min before cell lysis
|
| Treatment protocol |
Expression of DHFR or b2-DHFR was induced for 4.5 h by addition of 0.5% galactose. For replicate 1, cycloheximide was added to the cultures 2 min prior to harvesting.
|
| Growth protocol |
Cells were grown in minimal selective medium containing 2% lactate as sole carbon source
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Cells were harvested by fast filtration, flash-frozen in liquid nitrogen and lysed in a Retsch bead mill under cryogenic conditions. Cell powder was thawed and footprints were generated by RNase I digestion. Ribosomes were pelleted through a 25% (w/v) sucrose cushion by centrifugation for 20 min at 72,000 rpm in a Beckman TLA 100.2 rotor. RNA was extracted with hot acid phenol:chloroform.
Ribosome footprints were size selected by TBE-Urea PAGE. rRNA was removed with the Ribo-zero Gold kit (Illumina). RNA footprints were dephosphorylated, ligated to a linker sequence, reverse-transcribed to cDNA and circularized. Illumina sequencing barcodes were added by PCR amplification. Libraries were quantified with qPCR (Kapa Biosystems)
|
| |
|
| Library strategy |
OTHER |
| Library source |
transcriptomic |
| Library selection |
other |
| Instrument model |
Illumina HiSeq 4000 |
| |
|
| Description |
ribosome footprints
|
| Data processing |
Library strategy: Ribosome Profiling (Ribo-Seq)
Illumina CASAVA 1.8 was used for base calling and demultiplexing.
Adapter sequences were trimmed using Cutadapt v2.8
rRNA reads were removed by alignment with Bowtie v1.2.3
Remaining reads were aligned to a library of sequences of open reading frames containing 1000 nucleotides up- and downstream of the ORF, obtained from the yeast genome database SGD (genome release R64-2-1).
Each read was assigned to a specific A-site nucleotide by empirically determining a nucleotide offset from the 5' end of each fragment length that displayed three nucleotide periodicity.
Genome_build: R64-2-1
Supplementary_files_format_and_content: Density files contain the read counts for each gene along the sequence in tab-delimited format. Column 1: Systematic gene name; Column 2: Position relative to annotated Start of ORF (0 being the A from the start codon ATG); Column 3: Nucleotide at this position; Column 4: Read count
|
| |
|
| Submission date |
Apr 13, 2021 |
| Last update date |
Dec 16, 2021 |
| Contact name |
Felix Christian Boos |
| E-mail(s) |
fboos@stanford.edu
|
| Organization name |
Stanford University
|
| Department |
Genetics
|
| Street address |
300 Pasteur Drive
|
| City |
Stanford |
| State/province |
CA |
| ZIP/Postal code |
94305 |
| Country |
USA |
| |
|
| Platform ID |
GPL21656 |
| Series (1) |
| GSE172017 |
Regulation of translation under mitoprotein-induced stress in Saccharomyces cerevisiae |
|
| Relations |
| BioSample |
SAMN18736570 |
| SRA |
SRX10596194 |
