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Sample GSM7676897 Query DataSets for GSM7676897
Status Public on Sep 30, 2023
Title Donor 2 unstimulated BDCA3
Sample type SRA
 
Source name Blood
Organism Homo sapiens
Characteristics tissue: Blood
cell type: cDC1 (BDCA3+ DC)
treatment: None
Treatment protocol DCs were stimulated with: pRNA (15μg/ ml) and Hiltonol (10 μg/ml) for 16 hours.
Growth protocol Cells were obtained from aphaeresis of 5 different donors. Peripheral blood mononuclear cells (PBMCs) were isolated by using Ficoll density centrifugation (Lymphoprep; Axis-Shield PoC AS, Oslo, Anti-Human Lineage Cocktail 1 in FITC (LIN1) (BD Bioscience Pharmingen, San Jose, CA) containing antibodies for CD3, CD14, CD16, CD19, CD20, CD56 receptors, together with the anti-FITC conjugated magnetic microbeads of Miltenyi Biotec (Bergisch-Gladbach, Germany) were used to deplete the LIN1+ cell fraction, by following manufacturer’s instructions. Next, CDC1were further purified by sorting (flowcytometry) using anti–BDCA-3–APC combined with anti-HLA-DR-PE-Vio770 (Miltenyi Biotec) to a purity of 99.9%. DCs were cultured in X-VIVO-15 medium (Lonza, Basel, Switzerland) supplemented with 2% human serum (Sanquin).
Extracted molecule total RNA
Extraction protocol RNA was harvested using Trizol (Invitrogen, MA, USA), following the standard protocol. Quality control of the isolated RNA (concentration, RIN, 28S/18S and size) was performed with Agilent 2100 Bioanalyzer (Agilent Technologies, Santa Clara, USA).
After extraction of total RNA and treatment with with DNase I, Oligo(dT) are used to isolate mRNA. Mixed with the fragmentation buffer, the mRNA are fragmented. ThencDNAis synthesized using the mRNA fragments as templates. Short fragments are purified and resolved with EB buffer for end reparation and single nucleotide A (adenine) addition. After that, the short fragments are connected with adapters. The suitable fragments are selected for thePCRamplification. During the QC steps, Agilent 2100 Bioanaylzer and ABI StepOnePlus Real-TimePCRSystem are used in quantification and qualification of the sample library. Then the library is sequenced using Illumina HiSeq 4000 or other sequencer when necessary.
 
Library strategy RNA-Seq
Library source transcriptomic
Library selection cDNA
Instrument model Illumina HiSeq 4000
 
Data processing FASTQ files provided by BGI were mapped to the human genome version GRCH38 using hisat2 version 2.1.0 with parameter --phred64 and default parameters otherwise. Alignments were saved as BAM files.
Feature counts were extracted from BAM files using the Bioconductor package "Rsubread" version 2.4.3 (Liao et al, doi 10.1093/nar/gkz114)
Entrez IDs were converted to gene symbols using the Bioconductor packages "org.Hs.eg.db" version 3.12.0 and "annotate" version 1.68.0.
Assembly: hg38
Supplementary files format and content: counts.csv is a CSV file containing a matrix of feature counts per sample
 
Submission date Aug 02, 2023
Last update date Sep 30, 2023
Contact name Johannes Textor
E-mail(s) johannes.textor@radboudumc.nl
Organization name Radboud University Medical Center
Street address Geert Groteplein 26-28
City Nijmegen
ZIP/Postal code 6525 GA
Country Netherlands
 
Platform ID GPL20301
Series (2)
GSE239898 Hiltonol and pRNA stimuli induce an immune activating profile in the transcriptomics of cDC1s [RNA-seq]
GSE239899 Hiltonol and pRNA stimuli induce an immune activating profile in the transcriptomics of cDC1s
Relations
BioSample SAMN36811387
SRA SRX21228519

Supplementary data files not provided
SRA Run SelectorHelp
Raw data are available in SRA
Processed data are available on Series record

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