|
| Status |
Public on Jul 01, 2021 |
| Title |
beforeFACS_Kin_MinusATc_Rep3 |
| Sample type |
SRA |
| |
|
| Source name |
Saccharomyces cerevisiae transformed with Plasmid-encoded DNA barcodes
|
| Organism |
synthetic construct |
| Characteristics |
yeast strain: BY4743
treatment: Before FACS
grna_library: Protein kinase gRNA library
crispri_induction: No Atc
sequenced molecule: plasmid DNA
|
| Treatment protocol |
Bulk yeast populations were treated with 250ng/µl anhydrotetracycline (ATc) to induce gRNA expression and CRISPR interference and without ATc as reference in 2-4 replicates. Yeast populations were grown in exponential phase. For competitive growth assays, populations were diluted to OD600=0.005 and grown over 1.5-2 days at temperatures 23, 30 and 38°C. For thermotolerance assays, populations were diluted to OD600=0.3, exposed to a lethal heat shock (150 min at 50°C) and recovered without maintaining CRISPRi induction. For HSR activity screens, populations were diluted to OD600=0.3, exposed to 42°C for 5h to induce HSR, and sorted based on their cellular Hsp70 GFP reporter intensity. At least 250.000 cells in both the top and bottom 5% of populations were collected and recovered.
|
| Growth protocol |
design protocol: Guide RNA oligonucleotides were designed to target 161 TFs (retrieved from yetfasco.ccbr.utoronto.ca on 16/04/2014 with DNA-binding=1 and dubious=false; De Boer and Hughes, 2012) and 129 PKs (retrieved from yeastkinome.org on 16/04/2014; Breitkreutz et al., 2010), consisting of 885 and 668 gRNAs, respectively. Each gene was covered by up to six different gRNAs. Guide RNA oligonucleotides were integrated into the NotI site of the pRS416 dCas9-Mxi1 plasmid via Gibson Assembly, amplified in E. coli, and transformed into the S. cerevisiae BY4743 SSA1pr-GFP reporter strain.
Transformed yeast populations were constantly grown in sythetic complete uracil dropout medium (URA3 selection gene on CRISPRi plasmids).
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
Before and after selection procedures, plasmid DNA was extracted using FastPrep and glass beads to destroy the yeast cell wall in combination with standard extraction methods. The gRNA barcodes amplified from the one-copy-per-cell CEN-plasmids. Illumina sequencing was performed in paired-end and 75-100 base pairs read length on NextSeq500 machines with 15% PhiX spike-in.
DNA barcode libraries were generated by introducing standard Illumina P5 and P7 adapters and inline sample barcodes by PCR. Library preparation is otherwise performed according to standard Illumina protocols
|
| |
|
| Library strategy |
OTHER |
| Library source |
genomic |
| Library selection |
other |
| Instrument model |
Illumina NextSeq 500 |
| |
|
| Description |
PCR-amplified DNA barcodes
|
| Data processing |
Library strategy: CRISPR screen
Sequencing data was demultiplexed with Jemultiplexer with standard parameters and base call quality controlled with FastQC. Reads were trimmed and aligned to the FASTA of DNA barcode sequences (FASTA available on series record) with the Burrows-Wheeler algorithm to compute read counts.
Fold changes for gRNA barcodes and for genes were computed in R code using the edgeR package, although other read-count based packages can be used alternatively, such as limma or DeSeq2. Read counts were normalized by library size. Fitness fold changes were calculated as contrasts between +ATc and -ATc condition (+ATc/-ATc) of populations that both underwent competitive growth selection (fitness effect). Fold changes of the thermotolerance screen are ratios between +ATc samples after heat shock versus before heat shock (thermotolerance effect). Fold changes of the HSR screen denote ratios between +ATc samples sorted for high cellular GFP reporter intensity versus low cellular GFP reporter intensity (Heat shock response effect, based on activity of the Hsp70 Ssa1 chaperone promoter). Gene log2FCs were computed as mean log2FC of all gRNAs per gene. Alternatively, these can be computed without prior calculation of gRNA log2FCs by starting the edgeR analysis from the geometric mean of readcounts per gene. We further calculate rank-based gene score measures based on a subset of gRNAs with extreme effects and the median log2FC of gRNAs.
Supplementary_files_format_and_content: raw read counts and Log2 fold changes of gRNAs and genes
|
| |
|
| Submission date |
Jul 30, 2020 |
| Last update date |
Jul 01, 2021 |
| Contact name |
Cosimo Jann |
| Organization name |
EMBL Heidelberg
|
| Department |
Genome Biology
|
| Lab |
Group of Lars M. Steinmetz
|
| Street address |
Meyerhofstr. 1
|
| City |
Heidelberg |
| ZIP/Postal code |
69117 |
| Country |
Germany |
| |
|
| Platform ID |
GPL19424 |
| Series (1) |
| GSE155455 |
Dissecting the yeast heat shock response by CRISPRi/a |
|
| Relations |
| BioSample |
SAMN15680421 |
| SRA |
SRX8854714 |
