|
| Status |
Public on Apr 25, 2024 |
| Title |
wild-type RNA-seq, 3H, replicate 2 |
| Sample type |
SRA |
| |
|
| Source name |
Whole cells
|
| Organism |
Saccharomyces cerevisiae |
| Characteristics |
strain: wild-type (W303a)
hours in fe-deficient conditions: 3h
|
| Treatment protocol |
Yeast extracts were prepared by cryogrinding with BioSpec cryomill. Extracts were resuspended in buffer containging cycloheximide, and aliquots of cell lysates were used for footprint extraction and isolation of total RNA.
|
| Growth protocol |
Cells were grown in 500 mL of Kaiser SC media at 30°C for at least 15 hours to reach early exponential phase (OD600=0.2). Wild-type (W303a) and cth1Δcth2Δ mutant strains were treated with 100 μM of the Fe2+-specific chelator bathophenanthroline disulfonic acid (BPS) for 3h and 6h and collected using 0.45 µm PVDF membrane. Cells were flash frozen in liquid nitrogen and stored at -80C. As a control we collected wild-type (W303a) and cth1Δcth2Δ mutant cells that were grown in Fe-sufficient conditions (0h). Each sample was collected in triplicates (biological replicates) for each deletion strain and wild-type controls.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Lysates were subjected to ribosome footprinting by RNAse I treatment. After sucrose density gradient fractionation ribosome-protected fragments were purified from 80S peak fractions. In parallel, mRNA was isolated with Dynabeads mRNA Purification Kit (Life technologies) and subjected to alkaline hydrolysis. Fragments of 50-75 nt were used for library preparation.
The RNA-seq and Ribo-seq libraries were prepared using ARTseq Ribosome Profiling kit (Illumina)
|
| |
|
| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina NovaSeq 6000 |
| |
|
| Data processing |
De-multiplexing and base-calling was performed by Harvard University Bauer Center Sequencing Core
Sequence reads were trimmed for 3' adaptor sequence with cutadapt v.3.4
rRNA sequences were removed using alignment with bowtie v1.1.2
Sequence reads were mapped to the yeast genome with STAR 2.7.9a
Quantitation of reads was performed with Rsubread v.1.22.2
Genome_build: R64-2-1
Supplementary_files_format_and_content: Tab-delimited txt files with 3 columns: 1. SGD ORF name, 2. transcript length, 3. counts
|
| |
|
| Submission date |
Jan 04, 2022 |
| Last update date |
Apr 25, 2024 |
| Contact name |
Vyacheslav Labunskyy |
| E-mail(s) |
vlabuns@bu.edu
|
| Organization name |
Boston University School of Medicine
|
| Department |
Department of Dermatology
|
| Lab |
Labunskyy Lab
|
| Street address |
609 Albany St
|
| City |
Boston |
| State/province |
MA |
| ZIP/Postal code |
02118 |
| Country |
USA |
| |
|
| Platform ID |
GPL27812 |
| Series (1) |
| GSE193025 |
Ribosome profiling reveals the role of yeast RNA-binding proteins Cth1 and Cth2 in translational regulation |
|
| Relations |
| BioSample |
SAMN24611855 |
| SRA |
SRX13600564 |
