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Sample GSM5029516 Query DataSets for GSM5029516
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

Supplementary file Size Download File type/resource
GSM5029516_CRISPRi_screen_host_processed.tar.gz 2.8 Mb (ftp)(http) TAR
SRA Run SelectorHelp
Raw data are available in SRA
Processed data provided as supplementary file

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