Ncounter gene expression system

The NanoString nCounter gene expression system (NanoString Technologies, Seattle, WA, USA) is a multiplexed probe detection system, in which a probe library is constructed with two sequence‐specific probes for each gene of interest.50, 51 mRNA expression levels are quantified via direct digital detection without the need to reverse transcribe mRNA to cDNA or the amplification of the resulting cDNA by qRT‐PCR.50, 51 The NanoString nCounter gene expression system has similar sensitivity to qRT‐PCR, superior sensitivity compared with microarrays,50, 51 and exhibits higher sensitivity for low‐abundance transcripts versus RNA‐seq.52 nCounter Mouse PanCancer Panel (NanoString Technologies) was applied to assess osteogenic, immune, and cytokine gene expression in long bone (femur + tibia) marrow and whole livers. Hybridization of samples was carried out, and products were run on the nCounter preparation station according to the manufacturer's instructions. Data were collected via the nCounter digital analyzer and evaluated by nSolver Analysis Software v2.6 (NanoString Technologies). Data were normalized to the geometric means of spiked‐in positive controls and built‐in housekeeping genes. Absolute quantification of mRNA was reported as normalized mRNA counts as previously described.9, 13

Total RNA was isolated from HT-29 cells after 18 h treatment with 2DG, TRAIL or both using the standardized Trizol protocol described above. MicroRNA was enriched using the miRNeasy Mini Kit (Qiagen; 217004). RNA samples were evaluated as biological replicates ensuring that the RNA concentration was 100 ng/μl with a 260/280 greater than 1.9 and a 260/230 greater than 1.8. Analysis was performed using the NanoString nCounter gene expression system.^{34 (link)} Normalization of the raw data was performed by NanoString. The geometric mean of the top 100 expressed miRs was calculated for each assay. Using these means, a normalization factor was calculated for each assay to account for miRNA sample content variation. Subsequent replicate analysis demonstrated reproducibility between samples with R² values ranging from 0.92 to 0.98. The nCounter assay also included a set of six internal, positive control probes and eight negative control probes. The highest count detected from a negative control was 24, which was used as the limit of reliable detection of other miRs in the assay.
For kinase inhibitor screening, 8×10³ HT-29 cells were seeded and treated with 10 mM 2DG, 50 ng/ml TRAIL and compounds from the kinase inhibitor library (Enzo Life Sciences, Farmingdale, NY, USA; BML-2832).

Tumour samples were collected from NXS2-mCD19 animals on day 18 of the experiment. The samples were collected and enzymatically digested (Liberase, Roche) into single-cell suspensions. CD45⁺ cells were sorted using BD FACS Melody (BD Biosciences) and RNA was isolated from these cells (RNeasy Plus mini kit, Qiagen). Then, mRNA levels were directly measured using the Mouse-Pan cancer immune-oncology kit from NanoString nCounter gene expression system (NanoString). Differential expression analyses of mRNA were performed using nSolver analysis software (NanoString) and visualized by ClustVis^{39 (link)}. Gene Ontology (GO) annotation analysis of the target genes was performed using the Metascape tool (http://metascape.org), which facilitates enrichment analysis of biological processes and pathways of input genes^{40 (link)}. Only genes upregulated more than 2 times in the CAR(NAP) T-cell group compared with the CAR T-cell group were imported into Metascape and output P value cut-off was set to P < 0.0001. Cell type profiling analyses were performed using nSolver analysis software (NanoString).