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| Status |
Public on Apr 05, 2022 |
| Title |
UninfectedGroup_T36 |
| Sample type |
SRA |
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| Source name |
Alveolar lavage fluid
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| Organism |
Sus scrofa |
| Characteristics |
source: Alveolar lavage fluid
group: Uninfected group
time point: 36 hpi
sequence library: Sample index 1
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| Extracted molecule |
total RNA |
| Extraction protocol |
Primary porcine alveolar macrophages (PAMs) were collected from 3-week-old Specific Pathogen Free (SPF) pigs, and maintained in RPMI 1640 medium (GIBCO, USA) supplemented with 10% fetal bovine serum (FBS) (GIBCO, USA), 200 mg/mL streptomycin, and 200 IU/mL penicillin at 37℃. The ASFV Pig/SY18 strain was isolated and further propagated in PAMs for amplification culture. Viral titration was performed by hemadsorption (HAD) assays and expressed as HAD 50 /ml. All experiments with live virus were conducted in biosafety level 3 (BSL-3) facilities in Harbin Veterinary Research Institute (HVRI) of the Chinese Academy of Agricultural Science (CAAS).
5×105 PAM cells per well were seeded in 12-well plate and infections were carried out at a multiplicity of infection (moi) of 1. After 1 h of incubation, the viruses were removed and the cells were washed twice by PBS, then fresh medium was added. At the indicated times (0h, 2h, 6h, 10h, 15h, 24h and 36h), cells were washed gently and trypsinized. Single cell suspensions were washed with PBS + 0.04%BSA, resuspended in 200 μL PBS + 0.04%BSA, passed through a 30 μm filter, and placed on ice. Cells were counted using trypan blue exclusion on a LUNA-II™ Automated Cell Counter (Logos Biosystems). The appropriate numbers of cells to achieve a targeted cell input of 10,000 cells per condition were used to generate the GEMs (Gel Bead-In Emulsion). The Chromium Next GEM Single Cell 3' GEM, Library & Gel Bead Kit v3.1(10X Genomics, Pleasanton, CA, USA) was used for GEMs generation, cDNA synthesis and library preparation following the manufacturer’s instruction.
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| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina NovaSeq 6000 |
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| Data processing |
Procession of the 10× gene expression data, including barcode processing and single-cell gene counting, was performed using software CellRanger (version: 3.0.2) (http://10xgenomics.com/), and then the sequenced data were mapped into manually combined reference genome with merging Ensembl sus scrofa genome (Sus_scrofa.Sscrofa11.1.dna_sm.toplevel.fa) and virus ASFV genome (MH766894_2.fa). Unique molecular identifiers (UMIs) were quantified based on manually combined annotation file of Ensembl sus scrofa GTF file (Sus_scrofa.Sscrofa11.1.94.gtf) and virus ASFV GTF file (MH766894_2.gtf).
Genome_build: Sus scrofa
Supplementary_files_format_and_content: "barcodes.tsv" files include barcode information, "features.tsv" files include gene features and "matrix.mtx" files include gene expression.
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| Submission date |
Mar 02, 2021 |
| Last update date |
Apr 05, 2022 |
| Contact name |
Yuxuan Zheng |
| Organization name |
Fudan University
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| Street address |
825 Zhangheng Rd.
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| City |
Shanghai |
| ZIP/Postal code |
201203 |
| Country |
China |
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| Platform ID |
GPL26351 |
| Series (1) |
| GSE168113 |
Transcriptome profiling in swine macrophages infected with African swine fever virus at single-cell resolution |
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| Relations |
| BioSample |
SAMN18114549 |
| SRA |
SRX10212290 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM5129164_UninfectedGroup_T36_barcodes.tsv.gz |
20.0 Mb |
(ftp)(http) |
TSV |
| GSM5129164_UninfectedGroup_T36_features.tsv.gz |
234.4 Kb |
(ftp)(http) |
TSV |
| GSM5129164_UninfectedGroup_T36_matrix.mtx.gz |
170.9 Mb |
(ftp)(http) |
MTX |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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