Ncounter sprint

The nCounter Human v3 miRNA panel (NanoString Technologies), which targets 799 miRNAs, was used for miRNA profiling according to the manufacturer’s instructions using 100 ng of total RNA from each sample and a hybridization time of 20 h. The hybridization products were then analyzed on the nCounter™ SPRINT (NanoString Technologies) platform. The raw data were processed using the nSOLVER 3.0 software (NanoString Technologies); first, a background subtraction was performed using the max of negative controls, and then positive control normalization was performed using the geometric mean of all positive controls. A second normalization was performed using the geometric mean of the best combination of any two miRs (hsa-let-7d-5p and hsa-miR-423-5p) as determined by the NormFinder algorithm [39 (link)], before exporting the data to Excel (Microsoft Corporation).

The nCounter Human v3 miRNA panel (NanoString Technologies), which target 799 miRNAs, was used for miRNA profiling. One hundred nanograms of total RNA from each sample was subjected to nCounter™ SPRINT (NanoString Technologies) analysis according to the manufacturer’s instructions. The raw data were processed using the nSOLVER 3.0 software (NanoString Technologies). Background subtraction and normalization using the geometric mean of all positive controls was performed as recommended by the manufacturer. A second normalization was performed using the geometric mean of the top 100 highest expressed miRNAs, before exporting the data to Excel (Microsoft Corporation).

The NanoString nCounter analyses were performed using a previously published custom-designed panel, in which ciRS-7 and circIKZF3 were included [59 (link)]. One hundred nanograms of total RNA was used and the hybridization was carried out for 20 hours before performing nCounter SPRINT (NanoString Technologies, Seattle, WA, USA) analyses according to the manufacturer’s instructions. The raw data were processed using the nSOLVER 4.0 software (NanoString Technologies). Background subtraction and normalization using the geometric mean of all positive controls was performed as recommended by the manufacturer. Finally, a second normalization using the geometric mean of the four most stable linear reference genes with reasonable expression levels (ACTB, ESD, SF3A1 and PUM1) was performed, before exporting the data to Excel (Microsoft Corporation).

The nCounter Human v3 miRNA panel (NanoString Technologies), which target 799 miRNAs, was used for miRNA profiling in the fractions of cancer- and stromal cells. One-hundred ng of total RNA from each sample was subjected to nCounter™ SPRINT (NanoString Technologies) analysis according to the manufacturer’s protocol using a 20 hour hybridization time. The raw data were processed using the nSOLVER 4.0 software (NanoString Technologies); first, a positive control normalization was performed using the geometric mean of all positive controls, then a second normalization using the geometric mean of the top 100 highest expressed targets was performed, before exporting the data to Excel (Microsoft Corporation, Redmond, WA, USA).

Cells were treated with either 6 μM cisplatin, 5 nM Docetaxel, or 25 nM Etoposide. Treated cells were filtered 1 day following release of chemotherapeutic treatment. Each population of filtered cells as well as nonfiltered untreated cells were lysed using a QIAshredder Kit (Qiagen) following the manufacturer’s protocol. RNA was extracted from lysates using an RNeasy Mini Kit (Qiagen) following the manufacturer’s protocol. RNA was converted to cDNA using the iScript cDNA Synthesis Kit (Bio-Rad) following the manufacturer’s protocol. The complete product was used as input for hybridization with 770 nCounter PanCancer Progression probes for 16 h according to manufacturer’s protocols. Loaded cartridges were run on an nCounter Sprint (NanoString Technologies). Gene expression data quality control was analyzed using nSolver Analysis Software 4.0.70 (NanoString Technologies). All samples were normalized to the total counts of the nCounter-defined positive controls to reduce lane-to-lane variation from cartridge loading and normalize binding affinity across all samples surveyed. mRNA transcript reads of less than 40 were considered undetected.

A custom CodeSet was designed to be used with the PlexSet Nanostring technology (Nanostring) to analyse the genes in Table S1. The mRNA from iPSC and BFCN samples was harvested in TriSURE (Bioline, #38032) and extracted following the manufacturer’s instructions. The mRNA concentration of the samples was measured using a Qubit 3.0 Fluorometer (Thermo Fisher Scientific). To determine the optimal amount of mRNA per sample needed to not saturate the Nanostring, a titration run was performed and analysed using the nSolver System (Nanostring). A total of 100 ng mRNA per sample were run in the nCounter SPRINT (Nanostring). To normalize the samples, a reference sample compiled of all the samples was run alongside each Probe Set, and a total of 10 housekeepers (AARS, ASB7, CCDC127, CNOT10, EID2, MTO1, RABEP2, SUPT7L, TADA2B, ZNF324B) were selected based on the literature and Nanostring recommendations. Samples generated were analysed for each gene independently showing the number of molecules counted by the nCounter SPRINT System after normalization by housekeepers using the nSolver System.