SAMPLE5_M2c MACROPHAGES
Homo sapiens
Protocols: Peripheral blood mononuclear cells (PBMCs) were isolated from buffy coats using Lymphoprep density gradient centrifugation. Monocytes were isolated by adherence to plastic and cultured in Iscove's modified Dulbecco's medium supplemented with 10% [v/v] heat-inactivated human pooled serum (HPS), penicillin (100 U/ml), streptomycin (100 ug/ml) and ciproxin (5 ug/ml) at 37C in a humidified atmosphere supplemented with 5% CO2. Monocytes were incubated for 5 days in the presence of different cytokines: IFN-gamma (250 U/ml) in combination with TNF-alpha (12.5 ng/ml) (M1), IL-4 (50 ng/ml) (M2a), IL-10 (50 ng/ml) (M2c) or medium alone. Total RNA was isolated from monocytes and 5-day cultured macrophages (unstimulated or cytokine-polarized), using TriPure Isolation Reagent (Roche) according to the manufacturers instructions. Library preparation was carried out following the SOLiD Total RNA-Seq Kit Protocol (PN 4445374, Life Technologies) according to the manufacturers protocol for Small RNA library preparation. Briefly, RNA samples were first enriched for their small RNA fraction, by size selection between 15nt and 40nt on 15% TBE-Urea acrylamide gels. The small RNA-enriched samples were hybridized and ligated for cDNA synthesis. cDNA libraries were further purified with the MinElute PCR Purification Kit (Qiagen). After a size-selection step on 10% TBE-Urea gels, cDNA samples were amplified on gel using 19 cycles instead of 15 recommended by the manufacturer. For multiplexing purposes, the cDNA samples were amplified using the SOLiD 3' Primers from the SOLiD RNA Barcoding Kit. cDNA libraries were purified using the PureLink PCR Micro Kit (Invitrogen) and subjected to a second round of size selection on gel and purification according to the manufacturers instructions. The quality of the cDNA library was assessed with a smear analysis using the Bioanalyzer 2100 software (Agilent).
ENA-FIRST-PUBLIC
2013-11-01T17:01:38Z
ENA-LAST-UPDATE
2018-03-08T16:48:35Z
External Id
SAMEA2224109
INSDC center name
Department of Experimental Immunology, Academic Medical Center of the University of Amterdam
INSDC first public
2013-11-01T17:01:38Z
INSDC last update
2018-03-08T16:48:35Z
INSDC status
public
Submitter Id
E-MTAB-1969:SAMPLE5_M2c MACROPHAGES
broker name
ArrayExpress
cell type
monocyte
common name
human
disease
normal
individual
Donor 1
sample name
E-MTAB-1969:SAMPLE5_M2c MACROPHAGES
scientific_name
Homo sapiens
specimen with known storage state
fresh specimen