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| Status |
Public on Jun 16, 2020 |
| Title |
MDCK_0_2_Run_2 |
| Sample type |
SRA |
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| Source name |
MDCK cells
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| Organism |
Canis lupus familiaris |
| Characteristics |
cell line: MDCK
virus: wtWF10, barcoded WF10 (mVar) and helper WF10
multiplicity of infection: 0.2
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| Treatment protocol |
In the first experiment, MDCK and DF-1 cells were infected with wildtype A/Guinea Fow/Hong Kong/WF10/99 (wtWF10) at a multiplicity of infection of 0.067, 0.2, 0.6 or 1.8. In the second experiment, MDCK and DF-1 cells were infected with the wtWF10 and a barcoded version of wtWF10 (mVar) at a 1:1 ratio that equated to a multiplicity of infection (MOI) of 0.02, 0.067, 0.2 and 0.6. A helper virus, another version of WF10, was added to cells at a final MOI of 1 for MDCK cells or 0.1 for DF-1 cells. Virus stocks were serially diluted in cold 1X PBS and incubated on ice until use. Viruses were titered using flow cytometry (using anti-NP antibody). Before infection, cells were washed three times with cold 1X PBS and placed on ice. To infect, cells were inoculated with a 200 µL volume inoculum (with respective MOIs) and placed on ice for 45 minutes. Plates were rocked back and forth every 10 minutes to evenly spread the inoculum and prevent the monolayers from drying. After 45 minutes, the inoculum was aspirated and 2 mL of pre-warmed (at 37ºC) infection media was added. Plates were incubated at 37ºC for 3 hours. Afterwards, the infection media was replaced with 2 mL of pre-warmed infection media supplemented with 1M NH4Cl. Plates were placed back into incubator for an additional 5 hours. Subsequently, culture media was aspirated and cells washed once with 1X PBS. Cells were then trypsinized with 200 µL of 0.25% Trypsin EDTA until all cells came off the plate and un-clumped. To each well, 0.5 mL of virus infection media was added and replicates were pooled (2 wells per MOI). Cells for each sample were counted. Samples were spun at 150 rcf for 3 minutes and washed with 0.5 mL of 1X PBS/0.04% BSA. Washings were performed two more times. Finally, cells were resuspended with 1X PBS/0.04% BSA to get a final cell count of 7 x 10e5 cells/mL for each sample.
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| Growth protocol |
DF-1 and MDCK cells were cultured in DMEM with 10% FBS and 1% pen/strep on 6-well culture plates at 5 x 10e5 cells per well and incubated at 5% CO2 and 37°C.
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| Extracted molecule |
total RNA |
| Extraction protocol |
Single-cell gel beads in emulsion (GEMs) generated by the 10x Genomics chromium controller were reverse transcribed followed by 14 cycles of cDNA amplification. After cDNA cleanup, quantity and quality control of the amplified cDNA were analyzed using Bioanalyzer. Libraries were constructed by fragmentation, end-repair and A-tailing followed by adaptor and sample index ligation according to the manufacturer’s instructions. Finally, library constructs were qualitatively analyzed by Bioanalyzer and quantified by q-PCR before sequencing.
Preparation for single-cell transcriptomic sequencing followed the protocol for 10x Genomics Chromium Single Cell platform.
10X Genomics 3` RNA-seq
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| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina NextSeq 500 |
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| Description |
Mammalian (dog) origin
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| Data processing |
Cell Ranger v2.2.0 mkfastq
Genome_build: The chicken genome uses the GRCg6a (Gallus_gallus.GRCg6a.dna.toplevel.fa.gz) genome assembly; the dog genome uses the CanFam3.1 (Canis_familiaris.CanFam3.1.dna.toplevel.fa.gz) genome assembly.
Supplementary_files_format_and_content: *tsv, *mtx: Tab-delimited files that either have each barcode on one 'axis' of the table or read counts for each gene on the other 'axis' of the table.
Supplementary_files_format_and_content: *fa, *gtf: Fasta and gtf files for reference genomes.
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| Submission date |
Oct 21, 2019 |
| Last update date |
Jun 16, 2020 |
| Contact name |
Gene Tan |
| E-mail(s) |
gtan@jcvi.org
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| Organization name |
J. Craig Venter Institute
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| Street address |
4120 Capricorn Lane
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| City |
La Joll |
| ZIP/Postal code |
92037 |
| Country |
USA |
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| Platform ID |
GPL21400 |
| Series (1) |
| GSE135553 |
Viral transcriptomic analysis of single cell-sorted of DF1 or MDCK cell lines infected with influenza A virus |
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| Relations |
| BioSample |
SAMN13072766 |
| SRA |
SRX7032077 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM4134184_MDCK_0_2_Run_2_barcodes.tsv.gz |
1.3 Kb |
(ftp)(http) |
TSV |
| GSM4134184_MDCK_0_2_Run_2_genes.tsv.gz |
141.6 Kb |
(ftp)(http) |
TSV |
| GSM4134184_MDCK_0_2_Run_2_matrix.mtx.gz |
2.4 Mb |
(ftp)(http) |
MTX |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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