NanoString analysis of neurones

Reporter Code Count (RCC) files were imported into nSolver™ Analysis Software 4.0 (NanoString Technologies, Seattle, WA, USA). The manufacturer’s recommended default parameters for quality control flagging were used for imaging (field of view registration, < 75%), binding density (< 0.1 or > 2.25), positive control linearity (R² value < 0.95), and positive control limit of detection (0.5 fM positive control ≤ 2 SD above the mean of the negative controls). The background threshold was calculated using the geometric mean of the negative controls. Each sample was first normalised to the geometric mean of the positive controls (with default flagging of normalisation factors, < 0.3 and > 3), followed by normalisation to the geometric mean of the housekeeping genes, ACTB, GAPDH, and RPLP0 (with default flagging of normalisation factors, < 0.1 and > 10). Differential expression was evaluated using the nCounter Advanced Analysis plugin version 2.0.115 (NanoString Technologies, Seattle, WA, USA). Statistical significance was determined using an unadjusted P < 0.05.