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Sample GSM3581420 Query DataSets for GSM3581420
Status Public on Feb 12, 2019
Title hDC + LPS_donor4
Sample type SRA
 
Source name Donor4
Organism Homo sapiens
Characteristics donor: Donor4
cell type: dendritic cells
Treatment protocol 6 hours exposure to 500 ng/ml LPS in the absence or presence of 5µM adenosine receptor 3 antagonist VUF 8504
Growth protocol Human peripheral blood mononuclear cells (PBMCs) were isolated from human buffy coats obtained from anonymized healthy donors using lymphocyte separation medium gradient centrifugation (Lymphoprep; Axis-Shield, Norway). Monocytes were isolated with anti-CD14 monoclonal antibody-coated Microbeads using MACS single-use separation columns from Miltenyi Biotec (Bergisch Gladbach, Germany) as described by the manufacturer. Purified CD14+ cells were resuspended in RPMI (Gibco - Thermo Fisher, Waltham, MA) containing 10% (v/v) FCS (Gibco) and penicillin 100 U/ml and streptomycin 0.1 mg/mL (Gibco), supplemented with ≥ 4 units recombinant human M-CSF/mL or ≥ 40 units recombinant human GM-CSF and 200 ng recombinant human IL-4/mL (all Peprotech) to yield macrophages or DC respectively. Half of the medium was replaced by fresh medium containing new growth factors every 3-4 days. After 6-7 days in culture cells were used for functional assays.
Extracted molecule total RNA
Extraction protocol Briefly, mRNA was isolated from total RNA using oligo-dT magnetic beads. After fragmentation of the mRNA, cDNA synthesis was performed followed by ligation of sequencing adapters and PCR amplification. The quality and yield after sample preparation was measured with a Fragment Analyzer (Advanced Analytical, Heidelberg, Germany).
The Ultra Directional RNA Library Prep Kit for Illumina (New England Biolabs, Ipswich, MA) was used to prepare and process the samples.
 
Library strategy RNA-Seq
Library source transcriptomic
Library selection cDNA
Instrument model Illumina NextSeq 500
 
Description 102727-001-007
Data processing The size of the resulting products was consistent with the expected size distribution (a broad peak between 300-500 bp, data not shown). Clustering and DNA sequencing using the Illumina NextSeq 500 was performed according to manufacturer's protocols. 1.6 pM of DNA was loaded and analyzed using NextSeq control software 2.0.2. Image analysis, base calling, and quality verification were performed with Illumina data analysis pipeline RTA v2.4.11 and Bcl2fastq v2.17. The reads were mapped to reference sequence Homo_sapiens.GCRh37.75 using a short read aligner based on Burrows-Wheeler transform (mismatch rate of max 2%). For the 16 samples the number of reads obtained was between 22537666 – 32979730. The % reads of reads aligned to the reference was between 95.4 – 95.7%. Reads that aligned to multiple locations were between 7.5 – 9.4%. Reads that aligned to genes were between 60.0 – 87.8%. Samtools 0.1.18 was used to sort and index the BAM files. The number of times a read aligned to a gene and normalized read counts in Fragments Per Kilobase Million were determined using in-house developed scripts. The R package DESeq 1.10.1 was used to calculate false discovery rate-corrected p-values for the genes differentially expressed in the tested samples.
Clustering and DNA sequencing using the Illumina NextSeq 500 was performed according to manufacturer's protocols. 1.6 pM of DNA was loaded and analyzed using NextSeq control software 2.0.2.
Image analysis, base calling, and quality verification were performed with Illumina data analysis pipeline RTA v2.4.11 and Bcl2fastq v2.17.
For the 16 samples the number of reads obtained was between 22537666 – 32979730. The % reads of reads aligned to the reference was between 95.4 – 95.7%. Reads that aligned to multiple locations were between 7.5 – 9.4%. Reads that aligned to genes were between 60.0 – 87.8%. Samtools 0.1.18 was used to sort and index the BAM files. The number of times a read aligned to a gene and normalized read counts in Fragments Per Kilobase Million were determined using in-house developed scripts. The R package DESeq 1.10.1 was used to calculate false discovery rate-corrected p-values for the genes differentially expressed in the tested samples.
Genome_build: The reads were mapped to reference sequence Homo_sapiens.GCRh37.75 using a short read aligner based on Burrows-Wheeler transform (mismatch rate of max 2%).
 
Submission date Jan 28, 2019
Last update date Feb 12, 2019
Contact name Jeffrey Bajramovic
E-mail(s) bajramovic@bprc.nl
Phone +31 15 2842722
Organization name Biomedical Primate Research Centre
Department Alternatives
Street address Lange Kleiweg 161
City Rijswijk
ZIP/Postal code 2288GJ
Country Netherlands
 
Platform ID GPL18573
Series (1)
GSE125747 The contribution of adenosine receptor 3-mediated signaling to TLR4-induced responses by human dendritic cells
Relations
BioSample SAMN10831101
SRA SRX5299805

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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