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Sample GSM8257670

Query DataSets for GSM8257670

Status

Public on Sep 05, 2024

Title

CL118_6

Sample type

SRA

Source name

dendritic cell

Organism

Homo sapiens

Characteristics

tissue: dendritic cell
blood donor: 118
treatment: MDDCs cultured with HTLV-1 infected cells and restimulated for additional 24h with LPS

Treatment protocol

Immature MDDCs (1.106 cells) were plated in 48-well plates and cocultured with HTLV-1-infected or control uninfected T-cells (2.105 cells) in 300µL complete MDDC medium supplemented with IL-4 and GM-CSF for 24h. After 24h, cells were harvested, or stimulated with LPS or medium for an additional 24h. Cells from MDDC / T-cell lines coculture were harvested and stained using biotin-coupled anti-CADM1 followed by AlexaFluor647-coupled streptavidin diluted in PBS 1X-1% BSA for 20 min at 4°C. After several washes in PBS, cells were then incubated with anti-AlexaFluor647 MicroBeads (Miltenyi Biotec, 130-091-395) and separated on MACS Separation LD Columns (Miltenyi Biotec, 130-042-901) according to the manufacturer’s instructions.

Growth protocol

MDDCs were derived from purified monocytes from healthy blood donors collected at Etablissement Français du Sang (EFS) from anonymous healthy blood donors according to the institutional Standard Operating Procedures for blood donation, including a signed informed consent. Blood was diluted in PBS 1X (Gibco) and peripheral blood mononuclear PBMCs were isolated using a density gradient separation using Ficoll-Paque. Monocytes were then isolated from PBMCs using a density gradient separation using Percoll Centrifugation Medium.Freshly or frozen monocytes were cultured in six-well plates at 3.106 cells/mL in complete MDDC medium: RPMI1640 GlutaMAX (Gibco) supplemented with 10% FCS, penicillin-streptomycin (100 U/mL and 100 µg/mL respectively), Hepes buffer (Gibco, 15630080; 10 mM), MEM non-essential amino acids (2,5mM, Gibco, 11140050), sodium pyruvate (Gibco, 11360070; 1 mM), and beta-mercaptoethanol (Gibco, 31350-010; 0.05 mM). MDDC medium was supplemented with IL-4 and GM-CSF (Miltenyi Biotec, 130-093-922 and 130-093-866; 100 ng/mL each) for differentiation. On day 3, the culture medium was refreshed by discarding half of the medium and adding the same volume of new MDDC medium and twice concentrated IL-4 and GM-CSF to all cell cultures. Immature MDDCs were harvested on day 5 or 6.
HTLV-1 infected T-cell lines (C91-PL from Cellosaurus ref CVCL_0197, and C8166 ref ECACC 8805160) or control uninfected T-cell lines (Jurkat from ATCC ref ACC 282) were maintained at a cell density of 0.5.106 cells/mL in complete RPMI medium: RPMI1640 GlutaMAX supplemented with 10% fetal calf serum (FCS) and penicillin-streptomycin (100 U/mL and 100 µg/mL respectively).

Extracted molecule

total RNA

Extraction protocol

MDDCs present in the flowtrough were lysed and total RNA was extracted using the NucleoSpin RNA Mini kit for RNA purification (Macherey-Nagel, 740955) according to the manufacturer’s instructions
Ribosomal RNA was removed from total RNA, followed by ethanol precipitation. After fragmentation, the first strand cDNA was synthesized using random hexamer primers. During the second strand cDNA synthesis, dUTPs were replaced with dTTPs in the reaction buffer. The directional library was ready after end repair, A-tailing, adapter ligation, size selection, enzyme digestion, amplification, and purification. The library was checked with Qubit and real-time PCR for quantification and bioanalyzer for size distribution detection. Quantified libraries have been pooled and sequenced on Illumina platforms, according to effective library concentration and data amount required.

Library strategy

RNA-Seq

Library source

transcriptomic

Library selection

cDNA

Instrument model

Illumina HiSeq 2500

Data processing

RNAseq data were quality tested using fastqc, quality trimmed then quantified using Kallisto. Processed data correspond to raw read counts.
Assembly: hg19
Supplementary files format and content: raw read counts

Submission date

May 07, 2024

Last update date

Sep 05, 2024

Contact name

franck mortreux

E-mail(s)

franck.mortreux@ens-lyon.fr

Organization name

oncovirology and Biotherapies, UMR5239, LBMC

Street address

46 allee d'Italie