|
| Status |
Public on Jan 12, 2022 |
| Title |
Pex25BirA_2minCHX_5minBiotin_input |
| Sample type |
SRA |
| |
|
| Source name |
S288C
|
| Organism |
Saccharomyces cerevisiae |
| Characteristics |
strain/background: S288C
genotype/variation: pex25::PEX25-mVenus-BirA::HIS5 rpl16a::RPL16a-HA-TEV-AVI rpl16b::RPL16b-HA-TEV-AVI leu2<delta>0 met15<delta>0 ura3<delta>0
biotin induction duration: 5min
sample type: input
|
| Treatment protocol |
For harvesting, cells were treated with 100 µg/ml cycloheximide for 2 minutes prior to biotin induction with 10 nM biotin for the times shown.
|
| Growth protocol |
Yeast cells were grown in SD media containing 0.125 ng/ml D-biotin to OD600 .4-.6.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Yeast cells were harvested by filtration, flash frozen, and cryogenically pulverized in a Retsch mixer-mill.
Footprinting was performed with RNase I, monosomes purified on 10-50% sucrose gradients and biotinylated ribosomes purified on streptavidin dynabeads. RNA was extracted with Trizol and protected fragments gel purified. Ends were repaired with T4 PNK and ligated to a pre-adenylated linker with T4 RNA Ligase 2. Unligated linkers were enzymatically removed and ligated fragments were gel purified, reverse transcribed and gel purified. The cDNA was circularized then PCR amplified.
|
| |
|
| Library strategy |
OTHER |
| Library source |
transcriptomic |
| Library selection |
other |
| Instrument model |
Illumina HiSeq 4000 |
| |
|
| Description |
pex25::PEX25-mVenus-BirA::HIS5 rpl16a::RPL16a-HA-TEV-AVI rpl16b::RPL16b-HA-TEV-AVI leu2Δ0 met15Δ0 ura3Δ0
Ribosome-protected mRNA fragments.
|
| Data processing |
Library strategy: Ribo-seq
Base calls were called with Illumina CASAVA software.
3' adaptor was trimmed using fastx_clipper with -l 24 -a CTGTAGGCACCATCAAT.
5' nucleotide was removed using fastx_trimmer -f 2.
Reads were aligned to rRNA, tRNA and cloning oligos using Bowtie v1.1.2. Unaligned (filtered) reads were aligned to a genomic index (sacCer3) using Tophat v2.1.1 with the following parameters: --bowtie1 --read-mismatches 0 --no-novel-juncs. Only uniquely-mapped reads were used for subsequent analysis.
Each alignment was assigned a specific P-site nucleotide using a 15 nt offset from the 3' end of the read. Gene level counts and rpkm were calculated using plastid cs count using a positions file that masks regions to which reads cannot uniquely align. For rpkm calculations, all reads mapping to any annotated exon (transcript) were used for the denominator for total reads mapped.
Genome_build: sacCer3 (R64)
Supplementary_files_format_and_content: *txt: Tab-delimited text files containing count and rpkm data.
|
| |
|
| Submission date |
Apr 23, 2018 |
| Last update date |
Jan 12, 2022 |
| Contact name |
Jonathan S Weissman |
| Organization name |
Whitehead Institute for Biomedical Research, MIT
|
| Department |
HHMI, Biology
|
| Lab |
Weissman
|
| Street address |
455 Main St
|
| City |
Cambridge |
| State/province |
MA |
| ZIP/Postal code |
02142 |
| Country |
USA |
| |
|
| Platform ID |
GPL21656 |
| Series (1) |
| GSE113567 |
Peroxisome function relies on organelle-associated mRNA translation |
|
| Relations |
| BioSample |
SAMN08972787 |
| SRA |
SRX3989345 |
