|
| Status |
Public on Jan 22, 2021 |
| Title |
CRISPRn_perturb_virus_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 nuclease screen with droplet-based single-cell RNA-seq readout (Perturb-seq)
sgrna target organism: Homo sapiens, HCMV
|
| 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 |
total RNA |
| Extraction protocol |
HFFs expressing Cas9-BFP were lentivirally transduced with a virus+host-targeting Perturb-seq library (see paper), followed by puromycin selection. Cells were seeded at a density of 250,000 per well of a 12-well plate and infected with an MOI of 5.0 with no additional media change before harvest. Infection times were staggered so that all time points were harvested in parallel, and pooled, aiming for roughly equal cell numbers for each time point.
For each MOI, pools of ~10,000 cells were prepared for single-cell transcriptomics using one lane each of the Chromium Single Cell 3′ Gene Expression Solution v2 according to manufacturer’s instructions (10x Genomics). Barcodes encoding the experimental condition were PCR-amplified from the final library and sequenced as a 5 % spike-in as described (as described in Adamson et al., 2016).
|
| |
|
| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina HiSeq 4000 |
| |
|
| Description |
Human cytomegalovirus (strain Merlin)
Droplet-base single-cell RNA-seq with ~5% spike-in of a guide barcode (GBC) amplicon
RNA (poly-A mRNA and GBC)
|
| Data processing |
Cellranger v.2.0.1 (10x Genomics) and GBC calling as described in Adamson et al., Cell 2016.
Genome_build: Custom, based on GRCh37 (hg19) and the HCMV Merlin genome (NCBI NC_006273.2), see HCMV_merlin_annotation_gtf.tar.gz
Supplementary_files_format_and_content: Tar archive of the cellranger output files (1) cell barcodes. (2) genes. (3) sparse cell x gene matrix of expression values. (4) cell identities, i.e. sgRNA expressed in each cell, determined from the guide barcode amplicons, indicating the sgRNA target.
|
| |
|
| 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 |
SAMN17487860 |
| SRA |
SRX9913205 |
