Genechip human gene 2.0 st

Two previously published transcriptomics datasets comparing wounded to intact skin were reanalyzed for keratin expression using custom R scripts. Iglesias-Bartolome and colleagues collected serial punch biopsy specimens from the arm and buccal mucosa, extracted RNA from the samples, and performed RNA sequencing⁴⁵ . Unwounded skin was represented by the initial samples, and wounded skin was represented by follow-up samples taken from the same locations. We downloaded the expression data (Gene Expression Omnibus GSE97615) for the arm skin samples and plotted keratin transcript levels (log-transformed RPKM) as a heatmap. Statistical analysis was not performed. Ramirez and colleagues collected tissue samples from diabetic foot ulcers and non-ulcerated foot skin, extracted RNA from the samples, and profiled transcripts using Affymetrix GeneChip Human Gene 2.0 ST microarrays^{46 ,47} . We downloaded the expression data (Gene Expression Omnibus GSE80178) and used the “oligo” R package to import, annotate, and preprocess the microarray data, including background-correction and normalization using the RMA algorithm⁴⁸ . Keratin expression intensity values for wound and control samples were plotted as a heatmap, and statistical analysis was not performed.

Total RNA from SGC7901-Lenti-shRNA against CTSF cell lines and negative control SGC7901-Lenti-shRNA cell lines was isolated using TRIzol reagent (Invitrogen) according to the manufacturer’s instructions. RNA purity was determined using an ultraviolet and visible spectrophotometer (UNIC, Shanghai, P.R. China). Samples with RNA purity were used for GeneChip hybridization and scanning. Labeling, hybridization, and staining of these samples were performed according to the Eukaryotic Target Preparation protocol in the Affymetrix Technical Manual (701021 rev. 4; Affymetrix, Santa Clara, CA, USA). In summary, 1 μg of purified total RNA was used in the synthesis of cDNA, and the cDNA was purified using the GeneChip Sample Cleanup Module (Affymetrix). The purified cDNA was amplified to produce biotin-labeled cRNA using the GeneChip 3′IVT Express Kit (T7; Enzo Life Sciences, Farmingdale, NY, USA). Labeled cRNA was fragmented and hybridized to GeneChip Human Gene 2.0 ST according to the manufacturer’s protocol (Affymetrix). The hybridized gene chips were washed and stained for antibody amplification. The gene chips were then scanned using the Gene array Scanner 3000 7G (Affymetrix).

Non-del5q/5q- AML primary samples (n = 3) and BM-CD34⁺ HSPCs (n = 2) were FACS purified (CD45⁺CD33⁺ purity >98%) and cultured with 10 µM lenalidomide for 48h before global gene expression profiling (GEP) as described.²⁹ Total RNA was then extracted using a Maxwell® RSC simplyRNA Cells Kit in a Maxwell® RSC Instrument (Promega) and its quality checked in the Agilent 2100 Bioanalyzer. Total RNA samples were labeled with Cy3 using the Quick-Amp Labeling Kit and hybridized with the Gene Expression Hybridization Kit to the GeneChip Human Gene 2.0 ST (Affymetrix) following Manufacturer’s instructions. Hierarchical clustering of genes was performed with the one minus correlation metric and the unweighted average distance. Only genes showing >1.5-fold change expression and p-value<0.05 were considered differentially expressed and were subjected to gene ontology (GO) term analysis using Gorilla^30,31 publicly available at http://cbl-gorilla.cs.technion.ac.il. Microarray data were deposited in the public Gene Expression Omnibus database, accession number GSE106748.