Gene expression analysis was conducted on the NanoString nCounter gene expression platform (NanoString Technologies, Inc., Seattle, WA, USA). We used a mixed code set consisting of a 730-gene panel (PanCancer Pathway) related to 13 cancer-associated canonical pathways (MAPK, STAT, PI3K, RAS, Cell Cycle, Apoptosis, Hedgehog, Wnt, DNA Damage Control, Transcriptional Regulation, Chromatin Modification, and the large TGF-β family) and a custom 30-gene panel (Supplementary Table S1 ) including those from the T cell-inflamed gene expression profile described by Ayers et al. [15 (link)] and trophoblast tolerance genes. Depending on concentrations, hybridization with Human PanCancer Progression probes (NanoString Technologies, USA, #XT-CSO-PROG1-12) was performed using 78–200 ng RNA, according to manufacturer’s instructions. After 17–20 h of incubation at 65 °C, the samples were processed on a NanoString nCounter FLEX platform. Raw counts from individual digital molecular barcodes were normalized on 6 positive internal controls and 40 housekeeping genes using nSolver 4.0 analysis software (NanoString Technologies, Inc., Seattle, WA, USA). The background was estimated from blank wells and six negative internal controls and was removed from raw counts.
Nsolver 4.0 analysis software
Manufactured by NanoString
Sourced in United States
NSolver 4.0 Analysis Software is a data analysis tool designed for use with NanoString's nCounter Analysis System. The software provides automated analysis of gene expression data generated by the nCounter platform.
Automatically generated - may contain errors
Lab products found in correlation
Ncounter prep station, by NanoString (4 mentions)
Qubit 4 fluorometer, by Thermo Fisher Scientific (3 mentions)
Ncounter digital analyzer, by NanoString (3 mentions)
Ncounter gene expression platform, by NanoString (2 mentions)
High pure ffpe rna isolation kit, by Roche (2 mentions)
Rneasy mini kit, by Qiagen (2 mentions)
Ncounter master kit, by NanoString (2 mentions)
Ncounter pancancer immune profiling panel, by NanoString (2 mentions)
Ncounter platform, by NanoString (1 mentions)
Ncounter analysis, by NanoString (1 mentions)
Ncounter advanced analysis package, by NanoString (1 mentions)
Rna isolation kit, by Bio&Sell (1 mentions)
Quant it ribogreen rna reagent, by Thermo Fisher Scientific (1 mentions)
Ds 11 fx, by DeNovix (1 mentions)
Mouse cd8 til microbeads, by Miltenyi Biotec (1 mentions)
Nanodrop, by Thermo Fisher Scientific (1 mentions)
Automacs pro system, by Miltenyi Biotec (1 mentions)
Rlt lysis buffer, by Qiagen (1 mentions)
Reliaprep ffpe total rna miniprep system, by Promega (1 mentions)
Reporter codeset, by NanoString (1 mentions)
22 protocols using nsolver 4.0 analysis software
1
Comprehensive Gene Expression Analysis
2
Nanostring-based Gene Expression Analysis
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Gene expression analysis was performed on transfected NSCLC cell lines using the nCounter platform (NanoString Technologies, Seattle, WA, USA) according to the manufacturer’s instructions. Specifically, we used the Human PanCancer Progression Panel that includes 740 cancer genes involved in the tumor progression processes such as angiogenesis, extracellular matrix remodeling, epithelial-to-mesenchymal transition and metastasis, plus 30 internal reference controls. Briefly, 150 ng of total RNA were bound with a Reporter CodeSet and a Capture ProbeSet and then hybridized at 65 °C for 21 h. Samples were purified and then loaded onto the nCounter Cartridge by the nCounter Prep Station, and RNA was quantified by using the Digital nCounter Nanostring. RCC files deriving from counting process were first evaluated using nSolver 4.0 Analysis Software (Nanostring) performing a quality control through the fields of view count, binding density parameter and the eventual presence of any warning flags. Data were then analyzed using the Advanced Analysis 2.0 plug-in of nSolver system. Target genes which achieved ≥1.5 and ≤−1.5-fold change values and p-value < 0.05 were considered for analysis. Raw data are available in GEO (ID: GSE198957).
3
Transcriptional Profiling of Fibrosis and Innate Immunity
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NanoString nCounter analysis [NanoString Technologies] was performed at the Center for Molecular Medicine, Karolinska Institute, Stockholm, Sweden. For analysis of FFPE curls from ileum, differential gene expression was assessed using the Human Fibrosis V2 Panel [NanoString nCounter; 770 genes including ten housekeeping genes], in which a Panel Plus containing 30 genes of interest was added [Supplementary Table 1 ]. For analysis of peripheral blood CD14+ monocytes, differential gene expression was assessed using the Human Myeloid Innate Immunity V2 Panel [NanoString nCounter; 770 genes including 40 housekeeping genes]. Graphics and visualization of gene expression were done using nSolver 4.0 Analysis Software [NanoString Technologies].
4
Immune Gene Expression in Tumor-Bearing Mice
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Lung, heart, colon and kidney were harvested from mice in four groups: 1.) naïve, 2.) unvaccinated tumor only, 3.) vaccinated with CD24-Fc or 4.) vaccinated with IgG-Fc on day 30 after tumor cell inoculation. RNA was extracted and gene expression was directly measured via counts of corresponding mRNA in each sample using an nCounter murine AutoImmune Profiling Panel (NanoString, Seattle, WA, USA). For full details, see our previous publication (20 (link)). Briefly, 100 ng of high-quality total RNA was hybridized with reporter probes, and then biotinylated capture probes at 65°C for 16–18 hr before being placed into the nCounter Prep station in which samples were affixed to a cartridge. Cartridges were then read by the nCounter Digital Analyzer optical scanner. Further advanced immune-profiling analysis was performed using nSolver 4.0 analysis software with nCounter advanced analysis package (NanoString Technologies). Genes were grouped into 14 immune cell types and 35 immune functions according to the manufacturer’s designation (20 (link)).
5
Profiling FFPE Tumor RNA with NanoString
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The RNA of 22 tumors was obtained from FFPE tissue. Five 5-µm thick sections were cut from the FFPE blocks and, by following the manufacturer’s instructions, total RNA was obtained with the High Pure FFPE RNA Isolation Kit (Roche, Basel, Switzerland). RNA concentrations were measured using the Qubit 4 Fluorometer (Thermo Fisher Scientific, Waltham, MA, USA). The RNA samples with adequate concentrations were hybridized to the nCounter® PanCancer IO 360TM Gene Expression Panel (NanoString, Seattle, WA, USA), containing 770 genes, for 16 h using a thermocycler. The samples were transferred to the nCounter Prep Station (NanoString, Seattle, WA, USA) for further processing. The gene expression profiles of the samples were digitalized with the nCounter Digital Analyzer. The results were quantified using nSolver 4.0 Analysis Software (NanoString, Seattle, WA, USA).
6
Profiling Tumor Immune Landscape
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RNA was isolated from excised tumors after generation of single‐cell suspensions using an RNA isolation kit (Bio & Sell, Germany) according to the supplier's protocol. The RNA concentration of each sample was determined using fluorescent RNA labeling (Quant‐it RiboGreen RNA Reagent; Thermo Fisher, Germany) and a fluorometer (DS‐11 FX; DeNovix, Germany). Absolute concentrations were calculated against a standard curve. Subsequent gene expression analysis was performed using the NanoString nCounter technology, which directly evaluates RNA levels without prior complementary DNA synthesis. Briefly, 50 ng RNA per sample was hybridized for 24 h at 65 °C with reporter and capture ProbeSets of the nCounter PanCancer IO 360 Panel targeting 770 genes specifically designed for research in immuno‐oncology. Immediately after, samples were loaded into the nCounter SPRINT cartridge and processed by the NanoString nCounter system. Differential gene expression analysis was performed using the nSolver 4.0 analysis software (all NanoString Technologies, USA). Pathway enrichment analysis was evaluated using GSEA Software[89 , 90 ] and processed with Cytoscape.[91 ] Cell fractions were quantified from bulk tissue gene expression profiles using CIBERSORTx.[92 ]
7
Nanostring Gene Expression Validation
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RNA samples from the same tumors used for sequencing (NGS), and two further samples per group, were chosen for gene expression validation by nanostring. RNA concentrations measured by a Qubit 4 Fluorometer (Thermo Fisher Scientific, USA). RNA samples with adequate concentrations were hybridized to the customized nCounter® gene panel (NanoString, Redwood, CA, USA). The applied custom gene panel was composed of 134 genes identified by NGS as differentially expressed with the highest FC and lowest p values. Genes with no or deficient information according to the literature were excluded. Samples were transferred to the nCounter Prep Station for further processing. The gene expression profiles of the samples were digitized with the nCounter Digital Analyzer. Results were quantified using the nSolver 4.0 Analysis Software (NanoString, Redwood, CA, USA). Background was determined with synthetic negative probes provided by the Nanostring company, calculating the background level at maximum negative control count number.
8
Profiling CD8+ Tumor-Infiltrating T Cells
9
Profiling Immune Infiltrates in Murine Islets
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Analysis was done with the NanoString Technologies Pan-Immunology panel as per the manufacturer’s instructions. Briefly, 9000–45,000 CD45+ islet infiltrates were sorted from KO or WT BMT recipients and cell pellets were lysed in 33% RLT lysis buffer (QIAGEN). Up to 3.5 μl lysate was used directly in a 15 μl hybridization reaction which was kept at 65 °C for 24 h. The hybridization product was prepped and used with the digital analyzer nCounter MAX/FLEX system. The data were analyzed with nSolver4.0 analysis software (NanoString Technologies). Differentially expressed genes (P value <0.05 after FDR correction) were identified and pathways were analyzed with IPA and upstream regulator analysis (https://www.qiagenbioinformatics.com/products/ingenuity-pathway-analysis/ ).
10
Profiling Gene Expression in FFPE Samples
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Total RNA was isolated from 1‐mm cores of tumour‐rich areas of FFPE tissue blocks, which were selected from the most representative location, as described above, by homogenisation of each sample using the ReliaPrep™ FFPE Total RNA Miniprep System (Promega, Madison, WI, USA). Levels of mRNA were assessed for a custom gene panel, which included the 34 genes in ClearCode34, as reported previously [15 (link)]. Gene expression was quantified using an nCounter Digital Analyzer (NanoString Technologies Inc., Seattle, WA, USA), and raw counts were generated using nSolver™ 4.0 Analysis Software (NanoString Technologies Inc.). The NanoString data were corrected using positive and negative spike‐in controls and were normalised using five reference genes (C14orf166, RPLR1, SNRPD2, TBP, and ABCF1). To classify the ccA/ccB subtypes, unsupervised hierarchical clustering (median clustering and correlation distance) was performed using hclust in R for the NanoString data for the combined primary, RVI, and relapsed tumour samples [17 (link)].
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