|
| Status |
Public on Jul 18, 2022 |
| Title |
cth2Δ Ribo-seq, young, replicate 2 |
| Sample type |
SRA |
| |
|
| Source name |
Whole cells
|
| Organism |
Saccharomyces cerevisiae |
| Characteristics |
strain: cth2<delta> (UCC8773 derivative)
age: young
library type: ribosome profiling
|
| 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 |
Wild-type (MEP strain UCC8773) and cth2△ (UCC8773 derivative) cells were grown to OD(600)=0.2 in 20 mL YPD containing nourseothricin (100 ug/mL) and hygromycin B (300 ug/mL). For each strain, 300x106 cells were labeled with biotin EZ-Link Sulfo-NHS-LC-Biotin (5 mg/mL) for 30 min at room temperature. 1.5x10^8 labeled cells were added to 700 mL of cold YPD medium containing 1 uM 17β-estradiol and grown at 30C for 20 h. Then 300 mL of fresh YPD medium containing 1 uM 17β-estradiol was added, and cells were incubated for an additional 10 h (total 30 h incubation time). These cells were used to isolate replicatively aged (OLD) cells. For isolation of young (YNG) samples, cells were labeled with biotin and cultured under the same conditions for 2 h, allowing direct comparison between young and old cells. Repilcatively aged and young cells were isolated using Dynabeads Biotin Binder magnetic beads and flesh frozen in liquid nitrogen. At least 100x10^6 cells were used to make each of the RNA-seq and ribosome profiling libraries.
|
| 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 HiSeq 2000 |
| |
|
| 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 |
Nov 22, 2021 |
| Last update date |
Jul 18, 2022 |
| 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 |
GPL13821 |
| Series (1) |
| GSE189306 |
Deficiency of the RNA-binding protein Cth2 extends yeast replicative lifespan by alleviating its repressive effects on mitochondrial function |
|
| Relations |
| BioSample |
SAMN23382964 |
| SRA |
SRX13190103 |
