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Sample GSM4134184 Query DataSets for GSM4134184
Status Public on Jun 16, 2020
Title MDCK_0_2_Run_2
Sample type SRA
Source name MDCK cells
Organism Canis lupus familiaris
Characteristics cell line: MDCK
virus: wtWF10, barcoded WF10 (mVar) and helper WF10
multiplicity of infection: 0.2
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.
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.
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
Library strategy RNA-Seq
Library source transcriptomic
Library selection cDNA
Instrument model Illumina NextSeq 500
Description Mammalian (dog) origin
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.
Submission date Oct 21, 2019
Last update date Jun 16, 2020
Contact name Gene Tan
Organization name J. Craig Venter Institute
Street address 4120 Capricorn Lane
City La Joll
ZIP/Postal code 92037
Country USA
Platform ID GPL21400
Series (1)
GSE135553 Viral transcriptomic analysis of single cell-sorted of DF1 or MDCK cell lines infected with influenza A virus
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 SelectorHelp
Raw data are available in SRA
Processed data provided as supplementary file

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