Tissue samples were laser captured using the MMI CellCut Plus (Molecular Machines & Industries, Glattbrugg Switzerland) as described in Golubeva et al 2013. Laser microdissected cells were lysed using the lysis buffer RLT from Qiagen Micro RNAeasy Kit (Qiagen Gaithersburg, MD) and total RNA lysates were prepared for NanoStrings analysis (Geiss et al 2008). In vitro JygMC(A) GFP/Luc cells were cultured either as monolayers in plates or 3D spheres on ultra-low adherent plates (third generation spheres). Total RNA from tissue samples was extracted using TRIzol reagent according to the manufacturer's recommendations (Invitrogen, Carlsbad, CA). Total RNA from cells was isolated and purified using the RNeasy Mini Kit and subjected to DNAse treatment (Qiagen, Gaithersburg, MD) in accordance to manufacturer instructions). Following extraction, 1ug of total RNA was reverse transcribed using the RETROscript® kit (Ambion, Carlsbad, CA) in accordance with manufacturer's instructions.
Label
biotin
Label protocol
The assays were performed according to the manufacturer’s instructions (NanoStrings Technologies, Seattle, WA)
Hybridization protocol
The assays were performed according to the manufacturer’s instructions (NanoStrings Technologies, Seattle, WA)
Scan protocol
The assays were performed according to the manufacturer’s instructions (NanoStrings Technologies, Seattle, WA)
Description
Primary tumor
Data processing
Normalization was performed using the software nSolver version 1.1 (NanoStrings Technologies). The normalized counts were imported into the MultiExperiment Viewer Version 10.2 (MeV_4_8, http://www.tm4.org) software (Saeed et al 2003; 2006). The gene expression valuesProbe intensities were median-centered and log-transformed. Significant transcripts were selected using a t-test or the ANOVA statistical test with p <0.01 or 0.05. For clustering samples based on gene expression profile, we applied an unsupervised hierarchical clustering using Pearson correlation and average linkage.
