|
| Status |
Public on Jan 22, 2021 |
| Title |
CRISPRi_screen_host |
| Sample type |
SRA |
| |
|
| Source name |
Primary foreskin fibroblasts
|
| Organisms |
Homo sapiens; Human herpesvirus 5 strain Merlin |
| Characteristics |
cell line: HFF
experiment type: pooled CRISPR interference screen
sgrna target organism: Homo sapiens
|
| Treatment protocol |
HFFs were engineered with lentiviral vectors to express Cas9-BFP or dCas9-BFP-KRAB, followed by enrichment by FACS.
sgRNA libraries were also delivered on lentiviral vectors, followed by Puromycin selection.
Cells were infected with HCMV at MOIs indicated for each sample and harvested at defined time points for extraction of genomic DNA for extraction of the sgRNA cassette, or for droplet-based single-cell RNA-seq.
|
| Growth protocol |
Human foreskin fibroblasts (HFFs; CRL-1634) and HCMV (strain Merlin; VR-1590) were purchased from the American Tissue Culture Collection.
HFFs were cultured in DMEM, supplemented with 10 % FBS and penicillin-streptomycin.
HCMV stocks were expanded by two rounds of propagation on HFFs and titered by serial dilution.
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
The pooled screen was carried out at 500-1,000x coverage. A t0 sample was harvested and the remaining cells either passaged normally, or infected with HCMV at an MOI of 0.1. Infected flasks were washed with PBS and given fresh media at days 3 and 5 post infection to remove dead cells, and harvested at day 7. Genomic DNA was extracted and digested with MfeI to release a fragment containing the sgRNA cassette, followed by gel-based extraction.
The gel-purified DNA fragment was PCR-amplified and analyzed by deep sequencing as described (Horlbeck et al., 2016).
|
| |
|
| Library strategy |
OTHER |
| Library source |
genomic |
| Library selection |
other |
| Instrument model |
Illumina HiSeq 4000 |
| |
|
| Description |
Human cytomegalovirus (strain Merlin)
Genomic DNA fragment amplicon sequencing
DNA (sgRNA cassette amplicon)
|
| Data processing |
sgRNA counts were calculated as described (Horlbeck et al., 2016).
Raw count data were normalized for read depth and a small constant added to account for missing values. Phenotypes of individual sgRNAs were expressed as log2-transformed ratios of adjusted read counts between samples. We calculated the mean of all sgRNAs specific to each gene.
Genome_build: CRISPRi v2 (Horlbeck et al., 2016)
Supplementary_files_format_and_content: Tar archive of: (1) raw counts for each library element. (2) gene-level phenotypes.
|
| |
|
| Submission date |
Jan 21, 2021 |
| Last update date |
Jan 23, 2021 |
| Contact name |
Marco Yannic Hein |
| E-mail(s) |
marco.hein@maxperutzlabs.ac.at
|
| Organization name |
Medical University of Vienna
|
| Department |
Max Perutz Labs
|
| Street address |
Dr.-Bohr-Gasse 9
|
| City |
Vienna |
| ZIP/Postal code |
1030 |
| Country |
Austria |
| |
|
| Platform ID |
GPL29636 |
| Series (1) |
| GSE165291 |
Functional single-cell genomics of human cytomegalovirus infection |
|
| Relations |
| BioSample |
SAMN17487864 |
| SRA |
SRX9913201 |
