|
| Status |
Public on Jun 22, 2022 |
| Title |
VERO E6_T16A_SARS-CoV-2, IC90 |
| Sample type |
SRA |
| |
|
| Source name |
kidney epithelium cell line
|
| Organism |
Chlorocebus sabaeus |
| Characteristics |
cell line: VERO E6
crispr library: TKOv3
treatment: Infected w/ SARS-CoV-2 at IC90
growth medium: DMEM + 10% FBS +1% PenStrep
|
| Treatment protocol |
Cells that exhibited cytopathic effects (CPE) were infected at low MOI (Calu-3: 0.0005; Vero E6: 0.001, HEK293+A+T: 1) in serum free media for 1hr, virus inoculum was removed and replaced with regular culture media, after >80% CPE was achieved, surviving cells were maintained in standard media until confluence was reached. Cells that did not exhibit CPE were infected at MOI 1 Huh7 or MOI 10 (Caco-2) and were repeatedly exposed to fresh virus at every passage for 3 rounds.
|
| Growth protocol |
Cells were cultured in DMEM or EMEM media (as indicated) supplemented with 10% FBS and 1% Penicillin/Streptomycin and serially passaged for up to 15 doublings each, in triplicates.
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
Genomic DNA was extracted using the Wizard Genomic DNA Purification kit (Promega) or QIAamp DNA mini kit according to manufacturer’s instructions
For each sample, the gRNA cassette was amplified directly from genomic DNA using primers harboring Illumina TruSeq adaptors with i5 and i7 barcodes. The resulting sequencing libraries were pooled and gel-purified.
|
| |
|
| Library strategy |
OTHER |
| Library source |
genomic |
| Library selection |
other |
| Instrument model |
Illumina HiSeq 2500 |
| |
|
| Data processing |
Library strategy: CRISPRseq
FASTQ files were pre-processed to extract Cas9 guide sequence using a bespoke Perl script.
The Cas9 guide sequence was extracted from read1 files as the 20 bases preceeding 'CGGTGTTT'.
Trimmed FASTQ files were then aligned to the TKOv3 FASTA file using bowtie v0.12.1 using the following command line parameters: -p 16 -v 3 -l 18 --chunkmbs 256 -t <library_file> --un unmapped.fastq --al mapped.fastq -1 <trimmed_read1.fastq> aligned.sam
The number of reads aligning to each guide sequence was enumerated for each aligned .sam file, and alignment statistics were recorded. Guide IDs and counts were written to individual count files.
All individual count files were merged along with library annotations in R.
Supplementary_files_format_and_content: tab-delimited read count matrix
|
| |
|
| Submission date |
Jun 22, 2021 |
| Last update date |
Jun 22, 2022 |
| Contact name |
Jason Moffat |
| E-mail(s) |
j.moffat@utoronto.ca
|
| Organization name |
University of Toronto
|
| Department |
Molecular Genetics
|
| Street address |
160 College St
|
| City |
Toronto |
| State/province |
ON |
| ZIP/Postal code |
M5S 3E1 |
| Country |
Canada |
| |
|
| Platform ID |
GPL18770 |
| Series (1) |
| GSE178699 |
Systematic genome-scale identification of host factors for SARS-CoV-2 infection across models yields a core single gene dependency; ACE2 |
|
| Relations |
| BioSample |
SAMN19814427 |
| SRA |
SRX11199793 |
