Prior to antibody staining, blocking reagent, mouse IgG (1 mg/ml) was added to the melanoma or adult melanocyte cell suspension and incubated on ice for 10 min. All stainings were performed in 100 μl volume of cold HBSS containing 2 percent FBS. The following lineage antibodies were added: CD45, CD31, CD2, CD3, glycophorin A, EpCAM (all conjugated to pacific blue) plus an antibody against CD271 (Alexa Fluor647-conjugated) at a 1:50 dilution. Cells were incubated on ice in the dark for 30 min. After washing and centrifugation, cells were resuspended in 0.5 ml Hank's balanced salt solution (HBSS) containing 2% FBS and propidium iodide to allow exclusion of nonviable cells. CD271+ and CD271- cell populations were gated based on the isotype control background signal. Isolation of CD271+ and CD271- cells was achieved using a FACSAria III (BD Biosciences, San Jose, CA) cell sorter instrument.
Extracted molecule
total RNA
Extraction protocol
Total RNA was extracted from CD271+ and CD271- cell populations using Trizol Reagent (T9424-Millipore-Sigma).
Label
biotin
Label protocol
All RNA samples were processed using Ovation Pico WTA system V2.0 (NuGEN Technologies San Carlos, CA).
Hybridization protocol
All RNA samples hybridized to the Human Genome U133 Plus 2.0 microarray (Affymetrix, Santa Clara, CA) chips.
Scan protocol
Following hybridization and scanning fluorescent signals were obtained for 54675 probes that were further processed and mapped to 20535 gene-coding transcripts.
Data processing
The RMA oligo package was used for normalization and background correction of transcriptomic data. We utilized melanoma and melanocyte specimens sorted for CD271 expression status and subjected the data to unsupervised clustering analysis. CD271+ and CD271- specimens are denoted as CD271+ and CD271-, respectively. Both, rows (genes) and columns (specimens) were clustered using Pearson correlation distance and average linkage of log-transformed, normalized transcriptome array values. Pathway and enrichment analysis was performed by Ingenuity Pathway Analysis (IPA) software (QIAGEN Bioinformatics, Germany) and mapped with Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis. The enrichment study includes normalization of the enrichment score accounting for size of each gene set, yielding normalized enrichment score (NES). In addition to the calculated probability (p value) of hypothesis testing, there is adjustment for multiple hypothesis testing by controlling the proportion of false positives by calculating the false discovery rate (q values) corresponding to each NES, by comparing tails of the observed and the null distribution for the NES.
Submission date
Apr 24, 2019
Last update date
Jun 10, 2019
Contact name
Fabian Volker Filipp
Organization name
University of California Merced
Department
Systems Biology and Cancer Metabolism, Program for Quantitative Systems Biology
