title: Illumina HiSeq 2000 sequencing; Transcriptional profiling of primary human peripheral blood monocytes isolated from neurologically unaffected controls and treated in culture with LPS or vehicle.
design: Transcriptional profiling of primary human peripheral blood monocytes isolated from neurologically unaffected controls and treated in culture with LPS or vehicle.
library_construction_protocol: Human peripheral blood monocytes were isolated from fresh peripheral blood drawn with EDTA anticoagulation. Monocytes were purified by magnetic-activated cell sorting (MACS) with magnetic microbeads conjugated to anti-CD14 antibodies (positive selection) as per manufacturer's instructions (Miltenyi). Monocytes were cultured in RPMI1640 with 10% FCS and 1% penicillin/streptomycin supplementation at 37 °C in 5% CO2 humidified atmosphere on 24-well polystyrene cell culture plates. Peripheral blood monocytes were stimulated with cell-culture grade (\"BioXtra\"), γ-irradiated  lipopolysaccharides from Escherichia coli 0111:B4 (Sigma-Aldrich) dissolved in PBS to 1 µg/mL and diluted 1:1000 in cell-culture media to a final concentration of 1 ng/mL for 4 hours in technical duplicates. PBS was used as a vehicle control. 10^6 monocytes were lysed in situ with RLT+ buffer and genomic DNA contamination reduced with column-based genomic DNA removal as per suppliers' instructions (Qiagen). Total RNA was purified with the RNeasy Plus Kit as per manufacturer's instructions. Transcriptome libraries were prepared from 200 ng of total RNA with the TruSeq RNA Sample Preparation Kit v2, Set B (Illumina) as per supplier's instructions.