|
| Status |
Public on Nov 01, 2023 |
| Title |
15_WT_unstable_rep2 |
| Sample type |
SRA |
| |
|
| Source name |
ESM356-1 yeast strain transformed with the desired plasmid variants
|
| Organism |
Saccharomyces cerevisiae |
| Characteristics |
cell type: ESM356-1 yeast strain transformed with the desired plasmid variants
|
| Treatment protocol |
Yeast cells were sorted into 8 stability bins according to the tFT readout. The cells sorted into each bin were separately grown to saturation.
|
| Growth protocol |
Yeast cells were grown at 30°C in synthetic complete (SC) medium lacking histidine to log-phase with or without shifting to 37°C before sorting.
|
| Extracted molecule |
other |
| Extraction protocol |
Yeast cells were lysed by mechanical shearing and then the plasmids therein were isolated by column-purification.
The variable regions were PCR-amplified with the incorporation of unique barcodes for each stability bin. The PCR products were pooled proportionally according to the number of cells sorted into each bin, and then used as the template for another PCR for adding the sample barcodes and illumina sequencing adaptors.
|
| |
|
| Library strategy |
OTHER |
| Library source |
other |
| Library selection |
other |
| Instrument model |
Illumina NextSeq 500 |
| |
|
| Description |
tFT-X12 degron library in WT cells (replicate 2)
|
| Data processing |
Demultiplexed fastq files were checked for raw data quality and potential DNA contamination using FastQC (v0.11.9) and FastQScreen (v0.13) software.
Cutadapt (v4.0) was used to trim adapter sequences and for NextSeq-specific 3’ end quality trimming applying a threshold of 20.
Reads were filtered for a minimum length of 15 nucleotides and a maximum expected number of errors equal to 1 computed from quality values using Cutadapt (v4.0).
Read pairs were assembled using the paired-end read merger PEAR (v0.9.11) to reconstruct amplicon sequences based on a minimum of 8 bases overlap of the paired reads. Unassembled read pairs were discarded.
The variable region and the fraction barcode were subsequently extracted from the assembled sequences by applying a design-specific regular expression with the extract command in UMI-Tools (v1.1.2) using flanking common sequences.
Reads were counted for each observed combination of variable region and fraction barcode, and were exported as a count matrix in text format.
Protein stability indices (PSIs) per sequence variant were calculated from count data either per nucleotide sequence or per translated amino acid sequence as described (PMID 29727619) using the analysis pipeline available at: https://gitlab.rlp.net/imbforge/NGSpipe2go/-/tree/MPSprofiling
Assembly: Not applicable, because no alignment applied
Supplementary files format and content: tab delimited text files (tsv) containing counts of each observed combination of variable region (column ‘sequence’) and fraction barcode (column ‘id’).
Library strategy: Multiplexed protein stability (MPS) profiling using counts of amplicon DNA sequences.
|
| |
|
| Submission date |
Oct 27, 2023 |
| Last update date |
Nov 01, 2023 |
| Contact name |
Frank Rühle |
| E-mail(s) |
f.ruehle@imb-mainz.de
|
| Organization name |
Institute of Molecular Biology (IMB)
|
| Department |
Bioinformatics Core Facility
|
| Street address |
Ackermannweg 4
|
| City |
Mainz |
| ZIP/Postal code |
55128 |
| Country |
Germany |
| |
|
| Platform ID |
GPL19756 |
| Series (1) |
| GSE246422 |
Orphan quality control by an SCF ubiquitin ligase directed to pervasive C-degrons |
|
| Relations |
| BioSample |
SAMN38019602 |
| SRA |
SRX22250476 |
