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| Status |
Public on Mar 20, 2024 |
| Title |
p22086-s001_1-5-GEX |
| Sample type |
SRA |
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| Source name |
PBMC
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| Organism |
Homo sapiens |
| Characteristics |
tissue: PBMC
cell type: monocyte derived dendritic cells (moDCs)
library type: mRNA
antibodies/tags: none
infection: Mock
treatment: IFN-Beta
primer concentration: 0.1
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| Treatment protocol |
soluble human IFN-β treatment for 23 hrs
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| Growth protocol |
CD14+ monocytes were isolated from PMBCs of healthy human blood using the MojosortTM Human CD14 Selection Kit (BioLegend, Cat# 480026) To generate monocyte derived dendritic cells (moDCs), CD14+ monocytes were stimulated in non-tissue culture treated plates with 100ng/mL GM-CSF (R&D Systems, Cat# 7954-GM-020/CF) and IL-4 (R&D Systems, Cat# 6507-IL-025/CF) in complete RPMI. Complete RPMI was prepared as follows: RPMI 1640 (Corning, Cat# 10-040-CI) supplemented with 10% FBS, 2mM L-glutamine (Corning, Cat# 25-005-CI), 1mM HEPES (Corning, Cat# 25-060-CI), 1mM Sodium Pyruvate (Corning, Cat# 25-000-CI), 1x NEAA (Corning, Cat# 25-025-CI), and 1x Penicillin-Streptomycin. The next day, the media was replaced with fresh cytokines, removing un-adherent cells. After 4 more days in culture, differentiated moDCs consistently expressing CD14− CD11c+ HLA-DR+ DC-SIGN+ were harvested in the supernatant. For experiments, moDCs were cultured in complete RPMI in U-bottom 96-well plates at 1e5 cells per well.
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| Extracted molecule |
polyA RNA |
| Extraction protocol |
Cells were washed with 0.04% w/v bovine serum albumin (BSA) in PBS and counted.
Feature Barcoding was performed on moDCs prior to single cell RNAseq to label mock and ZIKV treated cells. Cells were blocked with Human TruStain FcX on ice for 10 minutes and then surface stained with BioLegend antibodies (TotalSeq™-C0253 anti-human Hashtag 3 Antibody and TotalSeq™-C0252 anti-human Hashtag 2 Antibody) on ice for 20 minutes.
The 10X Genomics Chromium Next GEM Single Cell 5’ v2 assay protocol (PN-1000264) was modified to be ZIKV-inclusive by adding a ZIKV-specific primer to the reverse transcription reaction mixture (Fig 1). All other steps were followed according to the manufacturer’s protocol. The ZIKV-specific primer (5’-AAGCAGTGGTATCAACGCAGAGTACCCTTCCACAAAGTCCCTATTGC-3’) was previously published by Lanciotti et al. (49) and modified to contain the non-poly-dT tag (underlined sequence) for cDNA amplification. After bead cleanup, cDNA was amplified 13 cycles, and gene expression (5’ GEX) and feature barcoded libraries were generated using the 5’ v2 Library Construction Kit (PN-1000190). 5’ GEX libraries were constructed using cDNA that was first fragmented at 32ºC for 5 min. Each sample was indexed with the Dual Index Kit TT Set A plate (PN-1000215) for 14 cycles of 98ºC for 20s, 54ºC for 30s, 72ºC for 20s, with a final extension of 72ºC for 1 min. Amplified feature barcoded libraries were constructed using DNA from supernatant cleanup. Each sample was indexed with the Dual Index Kit TN Set A plates (PN-1000250) for cycles of 98ºC for 20s, 54ºC for 30s, and 72ºC for 20s, with a final extension of 72ºC for 1 min. For QC, final libraries un on a HS DNA chip on an Agilent Bioanalyzer 2100. The 5’GEX and feature barcoded libraries were pooled separately and then combined at an appropriate ratio in order to obtain the targeted read depth of 50,000/cell and 5,000/cell for the 5’ GEX and feature barcoded libraries, respectively.
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| Library strategy |
RNA-Seq |
| Library source |
transcriptomic single cell |
| Library selection |
cDNA |
| Instrument model |
Illumina NovaSeq 6000 |
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| Description |
*features.tsv.gz files are available on the series record.
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| Data processing |
Composite host/virus references were created by concatenating sequence and annotation files for the host and virus and running cellranger mkref
Pre-processing of sequencing results to generate count matrices (gene expression, ADT and HTO barcode counts) was performed using the 10x genomics Cell Ranger pipeline
Further processing was done with Seurat v4.1 (cell and gene filtering, hashtag identification, clustering, differential gene expression analysis).
Assembly: KU501215.1 Zika virus strain PRVABC59
Assembly: ChlSab1.1.104
Assembly: GRCh38
Supplementary files format and content: 10x Genomics output files: barcodes.tsv.gz, features.tsv.gz, matrix.mtx.gz
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| Submission date |
Apr 25, 2023 |
| Last update date |
Mar 20, 2024 |
| Contact name |
Gregory K Tharp |
| E-mail(s) |
gktharp@emory.edu
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| Phone |
404-727-7797
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| Organization name |
Yerkes National Primate Research Center
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| Department |
Developmental and Cognitive Neuroscience
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| Lab |
Genomics Core
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| Street address |
954 Gatewood Dr
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| City |
Atlanta |
| State/province |
GA |
| ZIP/Postal code |
30329-4208 |
| Country |
USA |
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| Platform ID |
GPL24676 |
| Series (1) |
| GSE230571 |
Single-cell analysis reveals an antiviral network that controls Zika virus infection in human dendritic cells |
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| Relations |
| BioSample |
SAMN34369063 |
| SRA |
SRX20097304 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM7226852_p22086-s001_1-5-barcodes.tsv.gz |
26.9 Kb |
(ftp)(http) |
TSV |
| GSM7226852_p22086-s001_1-5-matrix.mtx.gz |
98.5 Mb |
(ftp)(http) |
MTX |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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