Nanodrop nd100

To conduct whole transcriptome profiling of monocytes, 20-mL of blood was drawn by antecubital venipuncture into Vacutainer Cell Preparation Tubes (Becton–Dickinson). After isolation of mononuclear cells through density-gradient centrifugation, monocytes were captured via immuno-magnetic positive selection with antibodies against CD14 (using reagents and an autoMACS Separator from Miltenyi Biotec), resulting in >90% purity by flow cytometry. Total RNA was extracted using RNAlater/RNeasy kits from Qiagen, and its quantity and integrity were verified using NanoDrop ND100 and Agilent BioAnalyzer instruments. 100 ng of RNA was converted into biotinylated cRNA target and hybridized to Illumina Human HT-12 v4.0 beadchips, then scanned on an Illumina iScan instrument at the UCLA Neuroscience Genomics Core. The raw data are deposited in Gene Expression Omnibus (Accession No. GSE52319).

RNA was harvested from cells in log phase growth using TRIzol (Invitrogen) and the RNeasy kit with DNase digestion (Qiagen) according to the manufacturer’s instructions. RNA was quantified using the NanoDrop ND-100 followed by quality assessment with the 2100 Bioanalyzer (Agilent Technologies). RNA concentrations for each sample was greater than 200ng/μl, with 28S/18S ratios greater than 2.2 and RNA integrity scores of 10 (10 scored as the highest). Sample amplification and labeling procedures were carried out using the Low RNA Input Fluorescent Linear Amplification Kit (Agilent Technologies). The labeled cRNA was purified using the RNeasy mini kit (Qiagen) and quantified. RNA spike-in controls (Agilent Technologies) were added to RNA samples before amplification. Samples (0.75 μg) labeled with Cy3 or Cy5 were mixed with control targets (Agilent Technologies), assembled on Oligo Microarray, hybridized and processed according to the Agilent microarray protocol. Scanning was performed with the Agilent G2505B microarray scanner using settings recommended by Agilent Technologies. Microarray data are available in the ArrayExpress database under accession number E-MTAB-1939.