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).
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