Ncounter digital analyzer optical scanner

RNA was purified from flow-sorted CD3⁺ or CD11b⁺ sorted cells using the RNEasyPlus micro kit, following the manufacturer’s instructions (Qiagen, Germantown, MD, USA). RNA samples were subsequently quantified and qualified using Nanodrop 1000 spectrophotometer (ThermoFisher, Waltham, MA, USA) and Bioanalyser Tape station (Agilent, Santa Clara, CA, USA) assays. The subsequent Nanostring analysis was performed at concentrations of 35ng/well and 25ng/well respectively for CD3⁺ cells and CD11b⁺ cells.
Samples were analyzed based on the nCounter® mouse PanCancer Immune profiling gene expression panel (NanoString Technologies, Seattle, WA, USA): Hybridation reaction was performed for 18h at 65°C. Fully automated nCounter FLEX analysis system; composed of an automated nCounter® Prep station and the nCounter® Digital Analyzer optical scanner (NanoString Technologies, Seattle, WA, USA) was used. Normalization was performed by using the geometric mean of the positive control counts as well as normalization genes present in the CodeSet Content: samples with normalization factors outside of the 0.3–3.0 range were excluded, samples with reference factors outside the 0.10–10.0 range were excluded as well. Gene expression analysis was performed using the nSolver v3.0 and Advanced analysis module softwares. (NanoString Technologies, Seattle, WA, USA).