|
| Status |
Public on Dec 30, 2016 |
| Title |
Sample 4_Ribosome footprints control rep2 |
| Sample type |
SRA |
| |
|
| Source name |
Ribosome footprints control
|
| Organism |
Saccharomyces cerevisiae |
| Characteristics |
strain: BY4741 POP5-TAP
plasmid: pTOWug2-836
medium: SC-Ura
|
| Treatment protocol |
The cell pellet was immediately immersed in a 50-mL conical tube filled with liquid nitrogen and 2 mL of lysis buffer [10 mM Tris-HCl (pH 7.0), 10 mM Tris-HCl (pH 8.0), 150 mM NaCl, 5 mM MgCl2, 1 mM DTT, 1% Triton X-100, 200 µg/mL cycloheximide, 25 U/mL Turbo DNase (Invitrogen)] was dripped into the tube.
|
| Growth protocol |
Yeast cells BY4741 expressing POP5-TAP from a single genomic locus and carrying pTOWug2-836 or pTOWug2-POP5 were grown in 150 mL of SC-Ura at 30 °C with vigorous shaking. These cells were grown from an initial OD600 of approximately 0.2 to OD600 around 0.7, and the cells were then harvested by vacuum filtration.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Extracts were prepared as previously described (Ingolia et al., 2009, Science, PMID: 19213877), except that the frozen cells were pulverized with a mixer mill at 30 Hz.
The total amount of RNA in the extracts was quantified using RiboGreen (Invitrogen), and then, 50 ng of total RNA was diluted to 300 µL with the lysis buffer. The sample was subjected to preparation of ribosome footprints according to a previously described method (Ingolia et al., 2012, Nat Protoc, PMID: 22836135). Briefly, total RNA was treated with RNase I (Epicentre), and then the ribosomal pellet was collected by sucrose cushion centrifugation. RNA was recovered from the pellet with TRIzol (Life Technologies) and purified with Direct-zol RNA MiniPrep (Zymo), followed by isopropanol precipitation. RNA-seq libraries were generated using TruSeq Standard Total RNA Library Prep Kit (Illumina) from total RNA prepared as described above.
|
| |
|
| Library strategy |
OTHER |
| Library source |
transcriptomic |
| Library selection |
other |
| Instrument model |
Illumina HiSeq 4000 |
| |
|
| Data processing |
library strategy: ribosome profiling
Basecalls performed using Illumina Casava 1.8 software
Removing adaptor sequences from library using fastx_clipper
Splitting reads by barcode using fastx-split
rRNA alignment using bowtie2 v2.0.6
Alignment of rRNA-depleted reads to yeast transcriptome using TopHat v2.0.7
Extracting mapped reads using samtools
Depleting duplicated reads using custom scripts
Read quantitation using custom scripts
Genome_build: sacCer3
Supplementary_files_format_and_content: Text files (*.txt) contain following information in three columns: (1) systematic gene names; (2) position of coding sequence, amino acid, used for counting read; (3) reads counts.
|
| |
|
| Submission date |
Aug 01, 2016 |
| Last update date |
May 15, 2019 |
| Contact name |
Hisao Moriya |
| E-mail(s) |
hisaom@cc.okayama-u.ac.jp
|
| Organization name |
Okayama University
|
| Department |
Research Core for Interdisciplinary Sciences
|
| Street address |
Tsushima-naka 3-1-1, Kita-Ku
|
| City |
Okayama |
| ZIP/Postal code |
700-8530 |
| Country |
Japan |
| |
|
| Platform ID |
GPL21656 |
| Series (1) |
| GSE85036 |
Post-Translational Dosage Compensation Buffers Genetic Perturbations to Stoichiometry of Protein Complexes |
|
| Relations |
| BioSample |
SAMN05468248 |
| SRA |
SRX1992968 |
