|
| Status |
Public on Nov 25, 2020 |
| Title |
ubi_∆ub3_rep1 |
| Sample type |
SRA |
| |
|
| Source name |
TLY61.A2
|
| Organism |
Saccharomyces cerevisiae |
| Characteristics |
genotype: As W303-1B but ubi3<delta>ub-HA::kanMX4
parental strain: W303
|
| Treatment protocol |
None. Cells were harvested at an OD600 of about 0.8
|
| Growth protocol |
The strains were grown in liquid YPD medium at 30 ºC
|
| Extracted molecule |
total RNA |
| Extraction protocol |
RNA was extracted from yeast cells using the hot phenol procedure. The amount of RNA was quantified in a Nanodrop 2000 Spectrophotometer (Thermo Scientific) and RNA integrity was checked by agarose gel electrophoresis.
HT-5Pseq libraries were prepared as in (Y. Zhang & V. Pelechano, 2020 bioRxiv) from 6 µg total RNA per sample. Briefly, the RNA was treated with TURBO DNase (Thermo Fisher) to digest DNA. After inactivation of the DNase, RNA was precipitated with ethanol and sodium acetate. The pellet was resuspended directly in ligation mix. A single-stranded RNA adapter was ligated to the RNA using T4 RNA ligase (NEB) o/n at 16 degrees C. The sample was purified with 2 volumes of RNAClean XP beads (Beckman Coulter). The purified RNA was reverse-transcribed using Superscript II (Thermo Fisher) with a combination of two custom primers, each containing a partial Illumina adapter sequence and either a random hexamer or an oligo-dT sequence. RNA was depleted with NaOH treatment at 65 degrees C. cDNA was purified using 2 volumes RNAClean XP beads (Beckman Coulter). Ribosomal cDNA was depleted using duplex-specific nuclease and specific probes. Remaining cDNA was purified using 2 volumes RNAClean XP beads (Beckman Coulter). Illumina sequencing libraries were prepared from the cDNA by PCR using Phusion high-fidelity PCR master mix (Thermo Fisher). After PCR, libraries were size-selected with 0.7 and 0.9 volumes AMPure XP beads.
|
| |
|
| Library strategy |
OTHER |
| Library source |
transcriptomic |
| Library selection |
other |
| Instrument model |
Illumina NextSeq 500 |
| |
|
| Description |
HT-5Pseq in ubi ∆ub3 mutant replicate 1
|
| Data processing |
Base calling and demultiplexing was performed using bcl2fastq and a sample sheet, with default settings
Illumina sequencing adapters were trimmed from the reads using cutadapt
The first 8 bases of each read were extracted as the UMI with umi-tools
Reads were mapped to the R64-1-1 version of S. cerevisiae genome using STAR aligner
Mapped files were deduplicated using umi-tools
Deduplicated files were processed with the fivepseq pipeline as of May 2020 (Nersisyan et al., https://doi.org/10.1101/2020.01.22.915421)
Deduplicated .bam files were converted to bedgraph using bedtools genomecov
Genome_build: R64-1-1
Supplementary_files_format_and_content: Strand-specific bedgraph files with coverage of only the 5' positions of reads across the genome
|
| |
|
| Submission date |
Jun 02, 2020 |
| Last update date |
Nov 26, 2020 |
| Contact name |
Vicent Pelechano |
| E-mail(s) |
vicente.pelechano.garcia@ki.se
|
| Organization name |
ScilifeLab - Karolinska Institutet
|
| Department |
MTC
|
| Street address |
Nobels väg 16
|
| City |
Solna |
| ZIP/Postal code |
SE-17177 |
| Country |
Sweden |
| |
|
| Platform ID |
GPL19756 |
| Series (1) |
| GSE151632 |
A functional connection between translation elongation and protein folding at the ribosome exit tunnel in Saccharomyces cerevisiae |
|
| Relations |
| BioSample |
SAMN15082108 |
| SRA |
SRX8453540 |
