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Sample GSM3581423

Query DataSets for GSM3581423

Status

Public on Feb 12, 2019

Title

hDC + VUF_donor5

Sample type

SRA

Source name

Donor5

Organism

Homo sapiens

Characteristics

donor: Donor5
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

102727-001-010

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