GSM5172819: LIB046507_TRA00182874_S13_quant; Homo sapiens; RNA-Seq
Prior to plating, the freshly isolated CD14+ cells were pelleted and lysed, and RNA isolation was performed using the Qiagen RNEasy Mini Kit. Likewise, RNA was isolated from liver stromal cell lines, previously plated at 10,000 cells per well 24hrs prior. This was performed following two rinses by gently- pipetted 200 uL volumes of sterile, room temperature PBS (Life Technologies). CD14+ cells were then plated alone, with stromal cell coculture, or in the presence of 500 pg/mL recombinant human IL6 (R&D Systems). Following 72 hours in culture, monocytes from each condition were harvested by pipetting and pelleted by centrifugation at 1000 RPM for 10 minutes. Stromal cell cultures adherent in the plate were rinsed twice with 200 uL volumes of sterile, room temperature PBS (Life Technologies). CD14+ Monocytes were re-purified using the CD14 MACS positive isolation kit (Milteyni) to minimize stromal cell contamination. RNA was prepared using the Qiagen RNEasy Mini Kit, according to manufacturer's instructions. Conditioned supernatant was pooled for each condition and frozen in sterile eppendorf tubes at -20°C prior to CBA analysis. Aliquots of RNA preparations were analyzed for quality and concentration at the Biopolymers facility at Harvard Medical School using the Agilent Bioanalyzer.